Ji Ling Mo, Pierre Devos, a n d G e r a r d Trausch
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
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
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.
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 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 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).
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).
To conclude, our results strongly suggest
that, assuming Na+ absorption in the posterior
gills proceeds via apical Na+ channels and basolateral Na'/K+-ATPase (Onken and Putzenlechner, 1995), a modification of Na'/K'-ATPase specific activity (through phosphorylation or dephosphorylation) is implicated in
some way in the cAMP-dependent stimulation effect of dopamine on Na+ transport
across the posterior gills of the Chinese crab
Eriocheir sinensis acclimated to fresh water.
This work was partially supported by a research grant
to Mo Ji Ling from the Facult6s Universitaires Notre-Dame
de La Paix (FUNDP), Namur, Belgium. We are thankful
to Dr. A. Pequeux for comments and advice on this manuscript and to Mrs. M.-C. Forget for technical assistance.
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RrcFtvFt�: 24 June 1997.
ACCEPTED: 17 October 1997.
Address: Laboratory of Comparative Biochemistry and
Physiology, Facult6s Universitaires Notre-Dame de La
Paix, B-5000, Namur, Belgium. (e-mail: [email protected]