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Comparative Biochemistry and Physiolo

Possible role of carbonic anhydrase, V–H+–ATPase, and Cl�/HCO3�

exchanger in electrogenic ion transport across the gills of the

euryhaline crab Chasmagnathus granulatus

G. Genovese a,b,*, N. Ortiz a, M.R. Urcola a, C.M. Luquet a,b

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

Ciudad Universitaria (C1428EHA) Buenos Aires, Argentinab CONICET (Consejo Nacional de Investigaciones Cientıficas y Tecnicas) Rivadavia 1917 (C1033AAJ) Buenos Aires, Argentina

Received 27 May 2005; received in revised form 26 August 2005; accepted 28 August 2005

Available online 27 September 2005

Abstract

We studied the participation of carbonic anhydrase (CA), V–H+–ATPase, and Cl�/HCO3� exchanger in electrogenic ion absorption through

the gills of Chasmagnathus granulatus. CA activity was measured in anterior gills and posterior gills after acclimation to 2�, 10�, 30� (about

seawater), and 45� salinity. The highest CA specific activity was detected in the microsomal fraction in anterior gills, and in the cytosolic

fraction, in posterior ones. Both fractions were strongly induced by decreasing salinity only in posterior gills. Perfusion of posterior gills from

crabs acclimated to either 2� or 10� with acetazolamide inhibited CA activity almost completely. In posterior gills from crabs acclimated to 2�

and perfused with 20� saline (iso-osmotic for these crabs), acetazolamide reduced transepithelial potential difference (Vte) by 47%, further

addition of ouabain enhanced the effect to 88%. Acetazolamide had no effect in the same gills perfused with 30� saline (iso-osmotic for seawater

acclimated crabs). Bafilomycin A1 and SITS (inhibitors of V–H+–ATPase and Cl�/HCO3�) reduced Vte by 15–16% in gills perfused with normal

20� saline, and by 77% and 45%, respectively when they were applied in Na-free 20� saline, suggesting the participation of those transporters

and cytosolic CA in electrogenic ion absorption.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Carbonic anhydrase; Chasmagnathus granulatus; Crab; Electrogenic ion transport; Gills; H+–ATPase; Cl�/HCO3� exchanger; Transepithelial potential

difference

1. Introduction

From their ancestral seawater habitat decapod crustaceans

have conquered brackish and freshwater environments

through estuarine systems. Estuaries, defined as bodies of

water in which freshwater mixes with salt water, possess a

longitudinal salinity gradient, which can be divided into

discrete zones on the basis of annual variation in salinity. A

great diversity of decapods is distributed along this gradient,

according to their degree of tolerance to the decreasing

salinity. Particularly, brachyuran crabs display a wide range of

tolerance to dilution of seawater, from the marine stenohaline

1095-6433/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.cbpa.2005.08.024

* Corresponding author. Departamento de Biodiversidad y Biologıa Expe-

rimental, FCEyN, UBA, Pabellon II, Ciudad Universitaria, (C1428EHA)

Buenos Aires, Argentina. Tel.: +54 11 4576 3348; fax: +54 11 4576 3384.

E-mail address: genovese@bg.fcen.uba.ar (G. Genovese).

species (such as Libinia emarginata and Cancer irroratus),

which can only inhabit the outer part of estuaries, to the

marine euryhaline or migratory species like the Chinese crab

Eriocheir sinensis and the blue crab Callinectes sapidus. E.

sinensis lives in very dilute or freshwater environments and

migrates towards estuaries and the sea for breeding, while C.

sapidus is an essentially marine species that may extend into

brackish and freshwater (Mantel and Farmer, 1983; Pequeux,

1995; Henry, 2001).

The range of euryhalinity of a given species strongly

depends on its capacity to ion- and osmo-regulate, that is, the

ability to maintain osmotic and ionic gradients between

hemolymph and water by means of active transport of ions,

especially across the gill epithelium. True estuarine species,

like the lesser blue crab Callinectes similis and the green shore

crab Carcinus maenas, are considered weak to moderately

strong hyper-regulators, since they are able to maintain limited

gy, Part A 142 (2005) 362 – 369

ww

G. Genovese et al. / Comparative Biochemistry and Physiology, Part A 142 (2005) 362–369 363

hemolymph to ambient gradients at low salinity, about 250 and

350 mosm kg�1 for C. similis and C. maenas, respectively

(Zanders, 1980; Piller et al., 1995; Henry et al., 1998). These

species are not able to inhabit the oligohaline zone of the

estuaries, where salinity is lower than 5�. On the other hand,

strong hyper-regulators, like C. sapidus and E. sinensis,

migrate between seawater and full freshwater, where they

maintain hemolymph-to-water gradients of more than 600

mosm kg�1 (Cameron, 1978; Onken, 1999; Henry, 2001).

The ion transport capacity that enables crab species to cope

with salinity variations has been often reported as depending

on the up-regulation of transport-related proteins, Na+/K+–

ATPase and carbonic anhydrase (CA) being the two most

studied. The former protein is known to provide a driving

force for ion uptake across the gills of all the euryhaline crabs

studied so far (see Pequeux, 1995; Onken and Riestenpatt,

1998; Henry, 2001; Morris, 2001; Lucu and Towle, 2003, for

reviews). In contrast, the principal functions of CA are much

more variable, depending on the studied species and the

enzyme’s subcellular location. CA reversibly catalyses the

conversion of CO2 and water to HCO3�and H+ and has

multiple locations within the cells. Particularly, the membrane

bound (microsomal) pool is believed to be involved in the

CO2 reactions that are part of transport and excretion process

(Henry and Swenson, 2000). Besides its respiratory role in

vertebrates erythrocytes (Henry and Swenson, 2000), the

cytosolic fraction of CA provides H+ and HCO3� as counter-

ions for Na+ and Cl� uptake in osmo-regulatory organs of

freshwater animals, e.g. gills of freshwater fishes and crabs,

and amphibian skin (see Pequeux, 1995 for a review; Harvey

and Wieczorek, 1997; Perry and Fryer, 1997; Sender et al.,

1999).

Several studies on freshwater acclimated Chinese crabs

indicated an important role of CA in electrogenic ion uptake,

by providing protons and bicarbonate ions to apical transport

proteins such as V-type H+–ATPase and Na+/H+ and Cl�/

HCO3� exchangers (Gilles and Pequeux, 1986, Onken et al.,

1991; Onken and Putzenlechner, 1995; Riestenpatt et al.,

1995). In agreement, induction of CA activity has been

detected in the gills of freshwater adapted Chinese crabs

(Olsowski et al., 1994) and also in the posterior gills of blue

crabs acclimated to low salinity (Henry and Cameron, 1982;

Piller et al., 1995; Henry, 2001). CA, V-type H+–ATPase and

Cl�/HCO3� exchangers, have recently been reported as being

involved in Cl� uptake through the gills of the fully freshwater

crab Dilocarcinus pagei (Onken and McNamara, 2002;

Weihrauch et al., 2004).

Chasmagnathus granulatus is a euryhaline hyper–hypo-

regulating crab (Mane-Garzon et al., 1974; Luquet et al., 1992;

Charmantier et al., 2002). Although it typically inhabits

estuarine environments of Brazil, Uruguay and Argentina, C.

granulatus differs from true estuarine species in its capacity to

colonise oligohaline habitats, like those found in the tributary

rivers of the Rıo de la Plata estuary, Argentina, where salinity is

about 0.5� at low tide (Botto and Irigoyen, 1979). Moreover,

individuals caught from salt marshes near the city of Rıo

Grande do Sul, Brazil, have tolerated exposure to freshwater,

regulating their osmotic and ionic concentrations (Bromberg et

al., 1995).

Previous reports have shown induction of CA activity in the

gills of C. granulatus upon acclimation to low salinity

(Monserrat et al., 1997; Lopez Mananes et al., 2000),

suggesting the involvement of this enzyme in ion regulation.

In a recent study on split gill lamellae mounted in a micro

Ussing chamber, Onken et al. (2003) have proposed that

electrogenic ion uptake across the gills of C. granulatus

proceeds in a similar way as in estuarine crabs like the green

shore crab (Riestenpatt et al., 1996), through apical Na+/2Cl�/

K+cotransporters, in series with Na+/K+–ATPase, K+ and Cl�

channels in the basolateral membrane. The former authors have

not found any change in short-circuit currents after addition of

acetazolamide, which suggests no involvement of CA in

electrogenic ion transport. This functional similarity with true

estuarine crabs is in contrast with the capacity of C. granulatus

to live in near freshwater conditions, producing hemolymph-to-

water gradients of up to 600 mosm kg�1 (Bromberg et al.,

1995; Charmantier et al., 2002), while estuarine species like the

green shore crab are restricted to media above 8� salinity and

produce much lower gradients.

Considering the strong osmo-regulatory capacity of C.

granulatus and previous results on increased CA activity in

the gills of this species at low salinities (Monserrat et al., 1997;

Lopez Mananes et al., 2000), we propose that acclimation of

this species to oligohaline medium triggers an additional ion-

uptake mechanism, based on apical V-type H+–ATPase,

soluble CA and an apical Cl�/HCO3� exchanger, which is

inhibited if the gill is perfused with a saline solution similar to

the hemolymph of seawater acclimated crabs. This hypothesis

predicts that inhibition of either of these transport-related

proteins will partially reduce the transepithelial potential

difference in the posterior gills of C. granulatus acclimated

to an oligohaline medium, e.g. 2� salinity. It can also be

predicted that crabs acclimated to oligohaline medium will

show increased carbonic anhydrase activity with respect to

those acclimated to estuarine conditions.

2. Materials and methods

2.1. Animals

Individuals of Chasmagnathus granulatus were collected

from Punta Rasa beach (36-18VS, 56-48VW), Buenos Aires

province, Argentina. In the laboratory, adult intermoult (Drach

and Tchernigovtzeff, 1967) male crabs (28–32 mm carapace

width) were randomly separated into four groups that were kept

in aquaria containing water of different salinities: 2�, 10�,

30�, and 45�. Water was prepared with artificial sea salts

(Marinemix, Marine Enterprises International Inc., USA) added

to dechlorinated tap water. During the 15-day acclimation

period the animals were fed twice a week with rabbit pellet

food and frozen fish (except for the 48 h before the

experiments), and water was changed in intervening days.

Before dissection, crabs were rapidly killed by destroying the

nervous system with a pair of scissors.

Table 1

Composition of the saline solutions used in the perfusion experiments

expressed as mmol L�1

Saline NaCl KCl MgCl2 CaCl2 HEPES NaHCO3 KHCO3 Choline C

20� 310 6.3 5 8.3 5 7 – –

30� 465 9.4 7.5 12.4 5 7 – –

20� Na-free – – 5 8.3 5 – 6.3 316

Glucose (2 mmol L�1) was added only to the perfusates.

G. Genovese et al. / Comparative Biochemistry and Physiology, Part A 142 (2005) 362–369364

2.2. Carbonic anhydrase (CA) activity

All the gills, except the first two, were removed from

selected crabs, placed in 1 :25 w/v ice cold buffer (225 mmol

L�1 mannitol, 75 mmol L�1 sucrose, 10 mmol L�1 Tris,

adjusted to a pH of 7.4 with 10% phosphoric acid), and

homogenised (1500 rpm, 20 strokes) in a glass tube with a

tight-fitting Teflon pestle similar to Schutt Labortechnik

(Gottingen, Germany) homogenizer cat. No 3.203.052 and

pestle cat. No 3.204.102. In part of the experiments, homo-

genates from anterior or posterior gills were pooled and

centrifuged at 1500 �g for 30 min (Sorvall RC5-C, USA)

and then centrifuged twice at 105,000 �g (Beckman XL90,

rotor 90ti, Fullerton, USA) for 1 h at 4 -C to separate

microsomal and cytosolic fractions. Mitochondria were not

pelleted before ultracentrifugation because a previous study on

gill Na+/K+–ATPase activity (Genovese et al., 2004) showed

that in this species most of the basolateral membrane sediments

together with mitochondria. Enzyme activity was measured as

follows: 400 AL of CO2-saturated water was added to a mixture

of 200 AL of gill homogenate and 5 mL of assay buffer at 4 -C,according to Henry (1991). The initial decrease in pH due to

the hydration of CO2 was recorded, using a Beckman A50 pH

meter. The change in pH represents the rate of CO2 hydration.

This change was measured within the same pH interval for all

the samples, during the first part of the experiment in which the

pH change was linear. Protein concentrations were determined

using Lowry et al. (1951). Specific enzyme activity was

calculated using the following formula (Burnett et al., 1981):

catalysed rate=uncatalysed rateð Þ � 1

mgprotein in sample¼ Umgprot�1

One unit (U) of CAwas defined as the concentration of enzyme

in the final assay volume necessary to obtain a ratio of two

between the catalysed rate and uncatalysed rate. For the

uncatalysed reaction the sample was replaced with 200 ALbuffer.

2.3. Gill perfusion

After removing the carapace, gills were gently excised and

placed in a Petri dish with saline solution. The afferent and

efferent vessels of gill 6 (representative of posterior gills) were

connected by polyethylene tubes (0.4 mm in diameter) to a

peristaltic pump (afferent) and to a glass tube (efferent).

Perfusion rate was kept at 0.1 mL min�1. The tubing was held

in position by an acrylic clamp and the preparation put into a

glass beaker with the appropriate saline solution, constantly

aerated.

2.4. Transepithelial potential difference (Vte)

Ag/AgCl electrodes were connected via agar bridges to the

external bath and to the glass tube collecting the perfusate

(internal side). Potential differences (outside–inside) were

recorded with a millivoltmeter (Metrix, Paris, France). In the

experiments with acetazolamide, after stabilization of Vte, each

gill was homogenised and stored at �20 -C until enzyme

activity assay was performed.

2.5. Saline solutions and drugs

Gills were perfused and bathed with 30� or 20� saline

adjusted with Tris base to the physiological pH of C.

granulatus, 7.75 (Luquet et al., 2002a) (Table 1). To inhibit

Na+/K+–ATPase and carbonic anhydrase, respectively, 5 mmol

L�1 ouabain and 0.2 mmol L�1 acetazolamide were dissolved

in the perfusates (Siebers et al., 1985; Onken et al., 1991).

Acetazolamide was solubilized by increasing pH up to 8.5 with

Tris base and then the saline was brought back to physiological

pH with HEPES (Luquet et al., 1998). For V–H+–ATPase

inhibition, 1 Amol L�1 bafilomycin A1 was added to the bath

solution previously dissolved in dimethylsulphoxide (DMSO)

(Onken and Putzenlechner, 1995). Salines with 0.1% DMSO

were tested as controls. SITS, an inhibitor of Cl�/HCO3�

exchanger, was directly dissolved in the bath solution at a final

concentration of 2 mmol L�1 (Onken et al., 1991). In a second

series of experiments posterior gills were symmetrically

perfused with 20� Na-free saline (NaCl replaced by choline

chloride) and treated with basolateral bafilomycin A1 or apical

SITS, 1 Amol L�1 and 2 mmol L�1, respectively.

2.6. Chemical reagents

All reagents were of analytical grade. NaCl, KCl, MgCl2,KHCO3 and glucose were purchased from Merck (Argentina).

CaCl2 and 4-2-hydroxyethyl-1-piperazineethanesulfonic acid

(HEPES) were obtained from JT Baker (USA), Choline

chloride was from Anedra (Argentina). Acetazolamide, man-

nitol, ouabain, phenylmethylsulphonyl fluoride (PMSF) and 4-

acetamido-4V-isothiocyanato-stilbene-2,2V-disulfonic acid (SITS)

were purchased from Sigma (USA). NaHCO3, phosphoric acid,

and sucrose were from Mallinckrodt (USA). Tris hydroxy-

methyl-aminomethane (Tris base) was obtained from Serva

(Germany). Bafilomycin A1 was from LC Labs. Dimethyl-

sulphoxide (DMSO) was from Carlo Erba Reagents (Italy).

2.7. Statistical analysis

For statistical analysis, gills were grouped into two groups

(anterior: 3–5 and posterior: 6–8) and repeated measures two-

way ANOVAwas performed. In other cases, one-way ANOVA

or paired Student t-test was performed when appropriate.

,

l

3 4 5 6 7 80

2

4

6

8

10

12

14

16

18

2010 ‰

30 ‰

45 ‰

Gill number

Spec

ific

CA

act

ivity

(U m

g pr

ot-1

)

Fig. 1. Specific activity of carbonic anhydrase (CA)TSE measured in gill

homogenates of Chasmagnathus granulatus acclimated to different salinities

(10�, 30�, and 45�, N =6).

2 10 300

5

10

15

20

25

30

35 a

b

c

Acclimation salinity (‰)

Spec

ific

CA

act

ivity

(U m

g pr

ot-1

)

Fig. 3. Differential induction of carbonic anhydrase activity (CA) in posterior

gill homogenates of Chasmagnathus granulatus acclimated to diluted salinities

(2� and 10�) and seawater (30�) (letters denote statistical differences with

p <0.05, N =8).

G. Genovese et al. / Comparative Biochemistry and Physiology, Part A 142 (2005) 362–369 365

Differences were considered significant where p <0.05 (Sokal

and Rohlf, 1981).

3. Results

Specific activity of CA measured in anterior gills (3–5)

was within the range 2.3–4.1 U mg prot�1. No significant

differences were detected among these gills at any of the

acclimation salinities (Fig. 1). Salinity had no effect on CA

activity from both fractions recovered after ultracentrifugation

of anterior gill homogenates (Fig. 2). In these gills the

microsomal fraction showed the highest specific activity

( p <0.001). Specific CA activity in posterior (6–8) gills

was 5 to 7-fold higher than in anterior gills ( p <0.05) (Fig. 1).

Furthermore, posterior gills from crabs acclimated to 10�

showed a two-fold higher enzyme activity than those from

seawater or 45� ( p <0.05). Specific activity of both

cytosolic and microsomal fractions from these gills was

strongly induced by decreasing salinity, the specific activity

cytosolic microsomal cytosolic microsomal0123456789

101112

Anterior gills Posterior gills

10 ‰

30 ‰

a

b

a

b*Spec

ific

CA

act

ivity

(U m

g pr

ot-1

)

Fig. 2. Specific activity of carbonic anhydrase (CA)TSE in microsomal and

cytosolic fractions of anterior and posterior gills of Chasmagnathus granulatus

acclimated to 10� and 30� (* represents comparisons with p <0.001 between

both fractions in anterior gills; a–b represents comparisons with p <0.001 in

posterior gills, N =4).

of the soluble form being between two and three-fold higher

than that of the membrane form ( p <0.001).

In order to test if CA activity was differentially induced

during acclimation to an oligohaline medium, crabs were

submitted to two hypo-osmotic media (2� and 10�), and

were also compared with crabs acclimated to seawater. There

was a significant increase of CA activity, from 18.6T0.9 at

10� to 30.1T2 U mg prot�1 at 2� (Fig. 3).

In preliminary electrophysiological experiments, there was

an increase in Vte after perfusion of posterior gills with

hypo-osmotic saline (20�) that was significantly reduced

with the addition of 0.2 mmol L�1 acetazolamide. The

inhibition with acetazolamide was detected in gills of crabs

acclimated at 2� but not in those taken from crabs

acclimated at 10� (data not shown). For testing whether

acetazolamide was equally inhibiting CA in both groups,

acetazolamide perfused gills and saline perfused gills

(control) were homogenised in buffer containing PMSF (a

protease inhibitor), 20 min after the gills had reached a

stable Vte and kept at �20 -C for further enzymatic assay.

Storing homogenates at �20 -C for 1 week did not affect

CA activity (data not shown). Fig. 4 shows a reversible

inhibition �90% of CA activity in both groups after

treatment with acetazolamide.

Control +Az -Az Control +Az 0

5

10

15

20

25

30a

b

a

b

2 ‰ acclimation salinity

10 ‰ acclimation salinity

Spec

ific

act

ivity

of

carb

onic

anh

ydra

se(U

mg

prot

-1)

Fig. 4. Specific activity of carbonic anhydrase (CA)TSE in posterior gills of

Chasmagnathus granulatus acclimated to 2� (hatched bars) or 10� (empty

bars), perfused with 0.2 mmol L�1 acetazolamide (+Az) (a–b, comparison

with p <0.01 between control and Az treatment in each salinity, N =5–7).

+DMSO +Bafilomycin0

1

2

3

4

5

6

b

a

20 ‰ perfusion saline

Vte

(mV

)

Fig. 6. Transepithelial potential difference (Vte) in isolated perfused posterio

gills of Chasmagnathus granulatus acclimated to 2�. Symmetrical perfusion

was carried out with saline 20�. Dimethylsulphoxide (0.1% DMSO) in the

bath produced a non-significant effect. Addition of 1 Amol l�1 bafilomycin A1

(inhibitor of V–H+–ATPase) in DMSO to the bath solution significantly

reduced the Vte (a –b, comparison with p <0.01, N =9).

G. Genovese et al. / Comparative Biochemistry and Physiology, Part A 142 (2005) 362–369366

In the light of these results, the possible ion transport role of

CA in posterior gills of crabs acclimated to 2� was further

studied. A shift in the perfusion and bath solutions from 30�

to 20� (hyposmotic-shock) produced a 200% augmentation of

Vte. Acetazolamide treatment reduced this increased Vte by

47T8%, and further addition of the Na+/K+–ATPase inhibitor

ouabain enhanced the inhibitory effect to 88T3% (Fig. 5A). In

a similar experiment, initial addition of ouabain caused a

reduction of 81T3% in Vte, and further addition of acetazol-

amide increased the inhibitory effect up to 92T3%. Effects of

both inhibitors were reversed by washing-out with normal

20� saline. When the latter protocol was followed but

perfusing the gills with 30� saline, ouabain caused a

73T6% reduction of V te while subsequent addition of

acetazolamide had a very small further effect, but not

significant (Fig. 5B).

To test if the effects of inhibiting CA on Vte were related to

the presence of an electrogenic proton pump, the specific

inhibitor of the V–H+–ATPase, bafilomycin A1 was applied to

posterior gills of crabs acclimated to 2�, symmetrically

perfused with 20� saline. Bafilomycin A1 in the bath

produced a significant 16T4% reduction in Vte that was hardly

washed-out (Fig. 6). DMSO alone (0.1%) did not affect Vte. In

a similar fashion, inhibition of a putative apical Cl�/HCO3�

exchanger with SITS caused a reversible reduction of Vte (Fig.

Ctrl Ctrl Az Az+Ou -Az-Ou0

1

2

3

4

5

6

20 ‰ 30 ‰30 ‰Perfusion saline

Vte

(mV

)V

te (m

V)

Ctrl Ctrl Ou Ou+Az -Ou-Az Ctrl Ou Ou+Az -Ou-Az0

1

2

3

4

5

6

20 ‰ 30 ‰30 ‰Perfusion saline

A

B

Fig. 5. Transepithelial potential difference (Vte) in isolated perfused posterior

gills of Chasmagnathus granulatus acclimated to 2� salinity. 5 mmol L�1

ouabain (Ou, inhibitor of Na+/K+–ATPase) and 0.2 mmol L�1 acetazolamide

(Az, inhibitor of CA) were applied basolaterally (N =5–6). (A) Az applied

before Ou. (B) Ou applied before Az, drugs were applied during symmetrical

perfusion first with 20� and second with 30� saline solution (Ctrl = Control).

Control +SITS -SITS0

1

3

4

5

6

7A

B

Ctrl 30 ‰

Ctrl 30 ‰

20 ‰ perfusion saline

ba a

Vte

(mV

)V

te (m

V)

Control Ou Ou+Sits -Ou-SITS0

1

3

4

5

6

7

**

20 ‰ perfusion saline

Fig. 7. Transepithelial potential difference (Vte) in isolated perfused posterio

gills of Chasmagnathus granulatus acclimated to 2�. (A) 2 mmol L�1 SITS

(inhibitor of Cl�/HCO3� exchanger) was applied in the bath solution (a–b

comparison with p <0.05, N =7). (B) 5 mmol L�1 Ouabain (Ou, inhibitor o

Na+/K+–ATPase) was applied basolaterally and further combined with 2 mmo

L�1 SITS (*, comparison with p <0.01 between treatments and control, N =5)

r

7A). When this drug was applied after ouabain, it enhanced the

inhibitory effect by 15T2%, (Fig. 7B).

Perfusion with 20� Na-free solution raised Vte to 16T4 mV

N =9, combining results of two experiments. Basolateral

r

,

f

l

.

Control +Bafilomycin -Bafilomycin

5

15

25

35A

B

a

bb

20 ‰ Na-free perfusion saline

20 ‰ Na-free perfusion saline

Vte

(mV

)V

te (m

V)

Control +SITS -SITS

2

4

6

8

10

12

b

a

b

Fig. 8. Transepithelial potential difference (V te) in isolated posterior gills of

Chasmagnathus granulatus acclimated to 2�, symmetrically perfused with

20� Na-free saline. (A) Basolateral addition of 1 Amol l�1 bafilomycin A1 in

0.1% DMSO (a–b, comparison with p <0.05, N =4). (B) 2 mmol L�1 SITS,

dissolved in the bath solution (a–b, comparison with p <0.05, N =5).

G. Genovese et al. / Comparative Biochemistry and Physiology, Part A 142 (2005) 362–369 367

addition of 1 Amol l�1 bafilomycin produced a mean inhibition

of 77T13% with maximal inhibition of 96% (Fig. 8A). In

similar conditions, 2 mmol L�1 SITS added to the bath

solution reduced Vte by 45T11%, with maximal inhibition of

78% (Fig. 8B).

4. Discussion

The distribution of CA among the gills of Chasmagnathus

granulatus reflects the specialization of gill functions previ-

ously described for this and other euryhaline species. We have

found in the anterior gills of C. granulatus, which are

predominantly respiratory (Genovese et al., 2000, 2004;

Luquet et al., 2000, 2002a,b) that most CA activity is

distributed in the membrane fraction and does not change with

salinity. This result agrees with numerous reports indicating an

important role of gill membrane-bound CA in respiration and

acid–base regulation, by converting bicarbonate ions from the

hemolymph to CO2, which is finally excreted by diffusion

(Henry, 1987, 1988; Bottcher and Siebers, 1993; Olsowski et

al., 1994; Bottcher et al., 1994b).

As it was previously reported for other osmo-regulating

crabs (Bottcher et al., 1990; Olsowski et al., 1994; Piller et al.,

1995; Henry et al., 2002), the CA activity of the posterior gills

of C. granulatus is several fold higher than that of anterior

gills, and is strongly induced by decreasing salinity, especially

when the medium becomes oligohaline (2�). Both cytosolic

and membrane-bound enzyme fractions are similarly induced,

in agreement with a previous report for the same crab by Lopez

Mananes et al. (2000). However we have found that the

cytosolic fraction is more abundant, while the latter authors

have detected higher activity in the membrane fraction. A

similar discrepancy can be found in the literature for

Callinectes sapidus, Carcinus maenas and Eriocheir sinensis

(Henry, 1988; Bottcher and Siebers, 1993; Bottcher et al.,

1994a,b; Olsowski et al., 1994; Henry et al., 2003).

An augmentation of cytosolic CA activity in C. granulatus

posterior gills can be related to increased production of protons

and bicarbonate as counterions for NaCl uptake. But, there is

no obvious relationship between the increased activity of

membrane-associated CA and the increased osmoregulatory

needs. A possible explanation is that the membrane fraction

obtained in these experiments includes mitochondria and thus

the increased CA activity could reflect increased mitochondrial

CA, related to higher energy expenditure.

Studies on isolated perfused gills (Bottcher et al., 1991) and

on split gill lamellae of C. maenas (Onken and Siebers, 1992)

have not shown any electrophysiological effect of the CA

inhibitor (acetazolamide), and concluded that in this species,

CA is mostly involved in carbon dioxide excretion and not in

osmo-regulation. This conclusion is in agreement with the

model proposed for ion uptake in estuarine crabs (Riestenpatt

et al., 1996; Onken and Riestenpatt, 1998), which does not

depend on the activity of CA.

In spite of the fact that almost complete inhibition of CA is

achieved with 0.2 mmol L�1 acetazolamide applied basolat-

erally to perfused gills 6 of C. granulatus acclimated to 10�,

no effect on transepithelial potential differences (Vte) can be

detected. Onken et al. (2003) working on split gill lamella of C.

granulatus acclimated to 2� have not found any difference in

short circuit current after acetazolamide treatment. But, it is

worth noting that the saline used in that study was 30� (iso-

osmotic to the hemolymph of crabs acclimated to 30�) and

not 20�, which is more similar to hemolymph of crabs

acclimated to 2� and stimulates the gill epithelium to absorb

ions (Tresguerres et al., 2003). In accordance, in posterior gills

of crabs acclimated to 2� salinity, perfused with 30� saline,

Vte is strongly reduced after inhibition of Na+/K+–ATPase with

ouabain but is not affected by acetazolamide. When the same

gills are perfused with 20� saline, they show a several-fold

higher Vte, which is significantly reduced by either ouabain or

acetazolamide. We infer that CA participates in electrogenic

ion transport when the gill epithelium is submitted to hypo-

osmotic stress.

Proton-motive forces generated by V–H+–ATPases have

been reported as important for osmo-regulation in two different

crab species: E. sinensis and Dilocarcinus pagei. It was

proposed that in those species that inhabit freshwater environ-

ments, apical V–H+–ATPase in parallel with a Cl�/HCO3�

exchanger are supplied with protons and bicarbonate ions by

G. Genovese et al. / Comparative Biochemistry and Physiology, Part A 142 (2005) 362–369368

CA (Onken et al., 1991, 1994; Onken and Putzenlechner, 1995;

Riestenpatt et al., 1995; Onken and McNamara, 2002;

Weihrauch et al., 2004). It is interesting to denote that in C.

maenas, which does not tolerate salinities below 8�, no

inhibition of Vte has been achieved using bafilomycin A1, a

specific inhibitor of the V–H+–ATPase, although this protein

can be detected widespread throughout the cytoplasm. It has

been concluded that in C. maenas V–H+–ATPase plays an

important role in acidification of intracellular organelles for

active ammonia excretion rather than in transbranchial NaCl

uptake (Weihrauch et al., 1998, 2001, 2002). In contrast,

inhibition of V–H+–ATPase with 1 Amol l�1 bafilomycin A1

added to the saline bathing posterior gills of C. granulatus,

results in a 16% inhibition of Vte. Consistently, inhibition of a

putative Cl�/HCO3� exchanger with SITS produces up to 15%

inhibition of Vte. The results of experiments in which the

transport inhibitors are added in the bath solution must be

considered with caution since the cuticle could be totally or

partially impermeable to some drugs (Riestenpatt et al., 1996)

or even these drugs could modify the cuticle conductance

(Onken and Riestenpatt, 2002). Moreover, due to the difficul-

ties derived from the low cuticular permeability, we have used

a high concentration of SITS (2 mmol L�1) which could have

inhibited any anion transporter.

In order to clarify the role of the V–H+–ATPase and the

Cl�/HCO3�, we have applied bafilomycin A1 and SITS under

hypo-osmotic Na-free conditions. At variance with Onken et al.

(2003) who reported almost total inhibition of short circuit

current in split gill lamellae of C. granulatus, superfused with

Na-free 30� saline, substitution of Na in our 20� saline (iso-

osmotic to the hemolymph of crabs acclimated to 2�)

increases Vte. Thus, under hypo-osmotic stress conditions the

posterior gills of this species produce a Na-independent Vte,

which is not evident at steady-state conditions (perfusion with

30� saline). The addition of bafilomycin to the Na-free

perfusate inhibits the Na-independent Vte by 77% while SITS

added to the bath in the same conditions reduces Vte by 45%.

Taken altogether, the results of these experiments suggest

that cytosolic CA provides bicarbonate for a Cl�/HCO3�

exchanger and protons for a V–H+–ATPase, in the apical

membrane of posterior gill ionocytes of C. granulatus

submitted to oligohaline medium. Under these conditions the

V–H+–ATPase seems to function as an additional energiser of

the gill epithelium, in addition to the Na+/K+–ATPase, which is

the major driving force in electrogenic NaCl transport across

the gills of this species (Luquet et al., 2002a, Onken et al.,

2003). As described previously for freshwater-tolerating crabs

(Onken and Putzenlechner, 1995; Onken and McNamara,

2002), the V–H+–ATPase could create an outwardly directed

bicarbonate gradient, which in turn energises the absorption of

chloride through an apical Cl�/HCO3� exchanger.

Acknowledgements

This work was supported by grants UBACyT X222, from

the University of Buenos Aires and PIP 2004 from CONICET

to C.M. Luquet, and a doctoral fellowship from CONICET to

G. Genovese. Material support was also obtained from Instituto

Antartico Argentino. We are indebted to Martın Tresguerres for

critical reading of the manuscript, Dr. Ines O’Farrell for

revising translation, and Dr. Marıa del Carmen Rıos, Mihaela

Senek, Gabriel Rosa and Daniel Medesani for their kind help.

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