Am J Physiol Regul Integr Comp Physiol 307: R634–R642, 2014.
First published July 30, 2014; doi:10.1152/ajpregu.00555.2013.
D1-like dopamine receptors downregulate Na⫹-K⫹-ATPase activity
and increase cAMP production in the posterior gills of the blue crab
Callinectes sapidus
Francis B. Arnaldo,1,2 Van Anthony M. Villar,2,3 Prasad R. Konkalmatt,3 Shaun A. Owens,2
Laureano D. Asico,2,3 John E. Jones,2,3 Jian Yang,3 Donald L. Lovett,4 Ines Armando,2,3 Pedro A. Jose,2,3,5
and Gisela P. Concepcion1
1
The Marine Science Institute, University of the Philippines, Diliman, Quezon City, Philippines; 2Department of Pediatrics,
Georgetown University School of Medicine, Washington, District of Columbia; 3Division of Nephrology, Department of
Medicine, University of Maryland School of Medicine, Baltimore, Maryland; 4Department of Biology, The College of New
Jersey, Ewing, New Jersey; and 5Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland
Submitted 18 December 2013; accepted in final form 9 July 2014
Arnaldo FB, Villar VM, Konkalmatt PR, Owens SA, Asico
LD, Jones JE, Yang J, Lovett DL, Armando I, Jose PA,
Concepcion GP. D1-like dopamine receptors downregulate Na⫹K⫹-ATPase activity and increase cAMP production in the posterior gills of the blue crab Callinectes sapidus. Am J Physiol Regul
Integr Comp Physiol 307: R634 –R642, 2014. First published July
30, 2014; doi:10.1152/ajpregu.00555.2013.—Dopamine-mediated
regulation of Na⫹-K⫹-ATPase activity in the posterior gills of some
crustaceans has been reported to be involved in osmoregulation. The
dopamine receptors of invertebrates are classified into three groups
based on their structure and pharmacology: D1- and D2-like receptors
and a distinct invertebrate receptor subtype (INDR). We tested the
hypothesis that a D1-like receptor is expressed in the blue crab
Callinectes sapidus and regulates Na⫹-K⫹-ATPase activity. RT-PCR,
using degenerate primers, showed the presence of D1R mRNA in the
posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1R) antibody in the
basolateral membrane and cytoplasm. Confocal microscopy showed
colocalization of Na⫹-K⫹-ATPase and D1R in the basolateral membrane. To determine the effect of D1-like receptor stimulation on
Na⫹-K⫹-ATPase activity, intact crabs acclimated to low salinity for 6
days were given an intracardiac infusion of the D1-like receptor
agonist fenoldopam, with or without the D1-like receptor antagonist
SCH23390. Fenoldopam increased cAMP production twofold and
decreased Na⫹-K⫹-ATPase activity by 50% in the posterior gills.
This effect was blocked by coinfusion with SCH23390, which had no
effect on Na⫹-K⫹-ATPase activity by itself. Fenoldopam minimally
decreased D1R protein expression (10%) but did not affect Na⫹K⫹-ATPase ␣-subunit protein expression. This study shows the presence of functional D1R in the posterior gills of euryhaline crabs
chronically exposed to low salinity and highlights the evolutionarily
conserved function of the dopamine receptors on sodium homeostasis.
dopamine receptor; Na⫹-K⫹-ATPase; blue crab; cAMP; posterior
gills
THE ATLANTIC BLUE CRAB,
Callinectes sapidus, a euryhaline
crustacean, must osmoregulate to survive in rapidly changing
saline environments of estuarine habitats. It is considered to be
a strong hyperosmoregulator that can maintain an almost constant hemolymph osmolality across a wide range of salinities
(8). The change in Na⫹-K⫹-ATPase activity in the posterior
gills, in response to changes in environmental osmolality, is
one of several osmoregulatory mechanisms in euryhaline crustaceans (25, 33, 39, 43, 44). However, the regulatory pathways
leading to modulation of Na⫹-K⫹-ATPase activity in posterior
gills of euryhaline crustaceans are not fully understood.
Dopamine has been shown to increase the Na⫹-K⫹-ATPase
activity in posterior gills of crustaceans through a cAMPdependent pathway (36). Invertebrates have three subfamilies
of dopamine receptors, i.e., 1) the DOP1 subfamily, which is
related to vertebrate D1-like receptors; 2) the INDR subfamily,
which is a distinct invertebrate group that functionally behaves
like vertebrate D1-like receptors; and 3) the invertebrate D2like receptor subfamily, which is related to vertebrate D2-like
receptors (38). Specifically in crustaceans, there are two types
of D1-like receptors, D1␣PAN and D1PAN (also termed D1R
and D5R in humans, respectively). D1-like receptors stimulate,
while D2-like receptors inhibit, adenylyl cyclase activity in
vertebrates and invertebrates (6, 14). D1-like receptors have
been pharmacologically characterized in the crustacean Eriocheir sinensis (36); however, the role of this receptor subtype
in the regulation of Na⫹-K⫹-ATPase in response to changes in
environmental osmolality is still unclear.
In mammals, there are currently two paradigms of the
D1-like dopamine receptor effect on ion transport that act in
opposite manner, depending on the cell type. In human lung
epithelia, dopamine via D1-like dopamine receptors increases
sodium transport by stimulating the rapid recruitment of Na⫹K⫹-ATPase from cellular endosomes to the basolateral membrane (5). In the proximal and distal tubules of the mammalian
kidney, however, dopamine decreases ion transport by acting
on D1-like dopamine receptors to increase cAMP, which leads
to the phosphorylation of Na⫹-K⫹-ATPase, resulting in its
internalization and inactivation (2, 4, 9, 11, 22, 26). Altered
arachidonic metabolism may result in the failure of dopamine
to inhibit Na⫹-K⫹-ATPase (28). The objective of the current
study was to test the hypothesis that D1-like receptors are
expressed in the posterior gills of the euryhaline blue crab C.
sapidus and function to increase cAMP production to ultimately regulate Na⫹-K⫹-ATPase activity.
MATERIALS AND METHODS
Address for reprint requests and other correspondence: P. A. Jose, Div.
of Nephrology, Dept. of Medicine, Univ. of Maryland School of Medicine,
20 Penn St., HSF II, Suite S003D, Baltimore, MD 21201 (e-mail:
pjose@medicine.umaryland.edu).
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Animals. Male blue crabs in intermolt were collected from the
Annapolis area and Hoopers Island, Chesapeake Bay, MD, between
June-October and housed at 25°C in filtered recirculating tanks
0363-6119/14 Copyright © 2014 the American Physiological Society
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D1-LIKE DA RECEPTORS INHIBIT THE BLUE CRAB SODIUM PUMP
containing dilute [10 parts per thousand (ppt) salinity] or full-strength
(32 ppt salinity) artificial seawater (Instant Ocean, Blacksburg, VA)
(20). Crabs weighed between 110 and 230 g and had carapace widths
from 11 to 15 cm. The crabs were fed once daily with a diet consisting
of processed oysters and dried pellet food. The crabs were exposed to
a 12:12-h light-dark photoperiod and, after exposure to dilute seawater
for 6 days, examined before experimentation. This duration of exposure was adequate to stimulate the hypoosmotic response in the crabs
and to upregulate expression of Na⫹-K⫹-ATPase in the epithelial
cells of the gills (33).
Drug infusion. Crabs undergoing drug infusion were removed from
the aerated tanks containing 10 ppt artificial seawater on day 5 of
acclimation (32, 33), and a 2-mm hole was drilled through the
carapace directly above the heart cavity, as described by Burnett et al.
(7). The drill-bit was pressed onto the carapace to create a depression
deep enough to allow needle-stick penetration but not cause any
bleeding. Latex rubber and cyanoacrylate adhesive were used to cover
the depression to prevent any hemolymph bleed out caused by the
puncture. The crabs were allowed to recover for 24 h before the study.
Subsequently, vehicle (137 mM NaCl, 3 mM KCl, 5 mM MgSO4, and
3 mM HEPES, pH 7.4) that is isosmotic with the crab’s hemolymph,
with or without drugs (1 M fenoldopam and 5 M SCH23390), was
infused directly (0.1 ml/min for 15 min) into the heart via an 18-gauge
needle connected to an infusion pump. Initial experiments using
lissamine green directly infused into the heart showed that the gills
were fully perfused within 5 min. The fenoldopam (1 M) and
SCH23390 (5 M) doses in our studies were based on studies in rats
in which the drugs were infused directly into the renal artery (18, 53).
These doses were lower than those used in the shore crab Chasmagnathus granulatus to avoid targeting other receptors, e.g., serotonin
receptors (12, 35), which may occur when higher doses are used. The
perfusion rate of 0.1 ml/min used was the same infusion rate used to
perfuse the gills of C. granulatus (21). A drug infusion period of 15
min was chosen because the D1-like receptor was phosphorylated and
internalized into the cell cytoplasm following 15 min of exposure to
dopamine in human embryonic kidney cells heterologously expressing the rat D1R (41).
Whole gill homogenate. The crabs were anesthetized by being put
in ice for 20 min and then killed by carapace removal. Gills 6 and 7
were excised to represent the posterior gills while gills 3 and 4 were
excised to represent the anterior gills. The gills were blotted dry and
homogenized in ice-cold buffer (250 mM sucrose, 2 mM EDTA, and
50 mM imidazole, pH 7.2) for Na⫹-K⫹-ATPase activity and cAMP
production assays or lysis buffer (1% Triton X, 0.1% SDS, and 0.5%
sodium deoxycholate) for immunoblotting studies. A protease inhibitor cocktail (10 mM AEBSF, 1 mM trypsin, and 10 mM PMSF) was
added to prevent proteolysis. Saponin (20 g/mg protein) was used to
permeabilize the membranes to maximize substrate accessibility for
the endogenous Na⫹-K⫹-ATPase. The crude homogenates were partially purified by centrifuging at 10,000 g. The final protein concentration (BCA kit; Pierce, Rockford, IL) of each supernatant was
adjusted to 1.0 –1.5 mg/ml before storage at ⫺80°C for subsequent
studies.
Immunoblotting. Samples of uniform amounts of protein were
resolved via 10% SDS-PAGE (Invitrogen, Carlsbad, CA). Proteins
were electrotransferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA) using a wet transfer apparatus (Invitrogen) and subjected
to immunoblotting, as reported previously (19, 52, 54). The primary
antibodies used were rabbit polyclonal anti-human D5R (Genetex, San
Antonio, TX), mouse monoclonal anti-chicken Na⫹-K⫹-ATPase
␣-subunit (developed by D. M. Fambrough and obtained from the
Developmental Studies Hybridoma Bank, The University of Iowa,
Department of Biological Sciences, Iowa City, IA), and mouse n
anti-actin (Sigma-Aldrich, St. Louis, MO). The rabbit anti-lobster
D1R antibody and immunogen were kindly provided by Dr. Deborah
J. Baro, Georgia State University. Donkey anti-mouse and goat
anti-rabbit secondary antibodies (Santa Cruz Biotechnology, Santa
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Cruz, CA) were used. The mouse monoclonal anti-chicken Na⫹-K⫹ATPase ␣-subunit antibody has been used successfully for a range of
arthropods, including C. sapidus (34, 48). The mouse monoclonal
anti-actin antibody is reactive to many species, including Drosophila.
The published immunogen for actin shares 71% sequence identity
with that of Drosophila and 79% with that of Callinectis.
The bands were visualized using an enhanced chemiluminescence
detection kit (Millipore, Billerica, MA) or by IR Western blot detection via the Odyssey Imager (Li-COR, Lincoln, NE) and quantified by
densitometric scan (Scion Images, Frederick, MD). Actin was used as
the housekeeping protein. To determine the D5R epitope specificity,
the immunoblots were incubated in antibody solution preincubated
with or without the blocking peptide (cat. no. GTX77969; Genetex,
San Antonio, TX). The company has not disclosed the immunogen
sequence but revealed that a pairwise alignment of the sequence of the
immunogen and that of Panulirus interruptus D1R (the closest
known homolog) showed 28% sequence identity. Total cell lysates
from HEK-293 cells heterologously expressing the human D5R and
total gill homogenates from the spiny lobster P. interruptus that were
supplemented with a protease inhibitor cocktail were used as positive
controls.
Degenerate primers and RT-PCR. Forward degenerate primers
were designed using the amino acid sequences YIHIKD (forward
primer: 5=-tayathcayathaargay-3=) of the D1-like receptors from the
seven arthropods phylogenetically closest to the blue crab. Of these
species, the crustacean P. interruptus is closest to the blue crab.
Amino acid sequence alignment of D1R from Apis mellifera
(NP_001011595), Bombyx mori (NP_001108459), Anopheles darling
(XP_315207), Ixodes scapularis (XP_002409287), Acyrthosiphon pisum (XP_001947683), P. interruptus (DQ295791), and Penaeus
monodon (JQ901712) showed that the sequence YIHIKD is highly
conserved in the D5R of these seven species. Four reverse primers of
20 –23 basepairs (23-bp reverse primer; 5=-GGGGTTTGTGGAGCTTGAGGCTG-3=) varying in their sizes at the 3=-end were derived
from the 100-bp nucleotide sequence of the D1R of the Celuca
pugilator (http://www.genome.ou.edu/FiddlerCrab_Illumina_seqs/
A4_B1_498828_100bp). RNA was isolated from the crab posterior
gills using the RNeasy mini kit (Qiagen, Valencia, CA), reverse
transcribed and amplified via PCR using four reverse primers mixed
in equimolar concentration (1 M) in combination with the forward
primer (1 M). The PCR products were resolved in 2% agarose gel
and visualized under ultraviolet light after staining with ethidium
bromide. The PCR amplicon was purified from the agarose gel and
cloned in to pCR4-TOPO cloning vector for further colony PCR and
sequence analyses.
Immunostaining and confocal microscopy. Crabs were acclimated
to 32 ppt salinity for 6 days before death and excision of gills 6 and
7. The gills were perfused with PBS then washed and stored in the
histological fixative HistoChoice (AMRESCO, Solon, OH) for 3 days
at 4°C. Four-millimeter-thick sections of the posterior gills were
prepared for microscopy. For immunohistochemistry, the sections
were incubated with either anti-D5R antibody or anti-Na⫹-K⫹-ATPase
␣-subunit antibody for 2 h at room temperature. After being washed
with PBS, the sections were incubated with donkey anti-mouse and
goat anti-rabbit secondary antibodies for 1 h at room temperature. The
biotinylated secondary antibodies were visualized using the ABC
complex kit (Pierce) and Vector VIP (Vector Laboratories, Burlingame, CA), counterstained with hematoxylin (Sigma-Aldrich), and
viewed with a Nikon E600 digital microscope.
For confocal microscopy, the slides were double immunostained
with rabbit anti-D5R antibody and mouse anti-Na⫹-K⫹-ATPase
␣-subunit antibody for 2 h at room temperature and probed with goat
anti-rabbit (H⫹L)-Alexa Fluor 488 (Molecular Probes, Carlsbad, CA)
and goat anti-mouse (H⫹L)-Alexa Fluor 568 (Molecular Probes)
antibodies for 30 min. After being washed with PBS twice and
distilled water once, the coverslips were mounted on glass slides using
Vectashield mounting medium and sealed with nail polish. Negative
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D1-LIKE DA RECEPTORS INHIBIT THE BLUE CRAB SODIUM PUMP
controls were likewise prepared but with the omission of the primary
antibodies. Colocalization of D1R and the Na⫹-K⫹-ATPase ␣-subunit was evaluated by laser scanning confocal microscopy, using an
Olympus Fluoview FV300 inverted microscope using 450-nm excitation and 535-nm emission filters for Alexa Fluor 488- and 560-nm
excitation and 645-nm emission filters for Alexa Fluor 568. Images
were overlaid using Olympus Fluoview FV300 version 3C Acquisition Software to determine colocalization.
cAMP assay. Whole gill homogenates were pipetted into plate
wells containing cAMP-specific rabbit antibody binding sites and
competitive cAMP-acetylcholinesterase (AChe) conjugate (Cayman
Chemical, Ann Arbor, MI). The plate was incubated overnight at 4°C
after which the wells were washed. The plate was developed with
Ellman’s reagent, which contains the AChe substrate, and incubated
in the dark for 2 h at room temperature. cAMP was quantified (412
nm) using a spectrophotometer (Thermofisher Scientific). cAMP content of the samples was calculated compared with known standards
and expressed as picomoles of cAMP per milligram of protein.
Measurement of Na⫹-K⫹-ATPase activity. Na⫹-K⫹-ATPase activity was measured (26) in an incubation buffer containing 140 mM
NaCl, 5 mM KCl, 5 mM MgCl2, 30 mM Tris·HCl, 1 mM EGTA, 3
mM Na2ATP, and [␥-32P]ATP (2–5 Ci/mmol) in tracer amounts (5
nCi/l), using a 15-min incubation period at 37°C in the presence or
absence of 2 mM ouabain, a Na⫹-K⫹-ATPase inhibitor. When
ouabain was present, NaCl and KCl were omitted from the incubation
buffer. The reaction was initiated by the addition of 10 l of homogenate to 90 l of incubation buffer. The reaction was terminated by
the addition of activated charcoal/trichloroacetic acid and rapid cooling on ice. After 1 h, the samples were centrifuged for 4 min at 14,000
rpm to separate the charcoal that contained the unhydrolyzed nucleotide. Radioactivity present in the supernatant containing the inorganic [␥-32P] produced by ATPase activity was measured in quadruplicate using a liquid scintillation spectrometer. Na⫹-K⫹-ATPase
activity was calculated by subtracting the ouabain-insensitive ATPase
activity from the total ATPase activity and expressed as millimoles of
Pi per milligram of protein per hour.
Chemicals. Fenoldopam bromide and SCH23390 were purchased
from Sigma-Aldrich. [␥-32P]ATP was purchased from Perkin-Elmer
Life Sciences. All other chemicals were obtained from Sigma-Aldrich.
Statistical analysis. Numerical data are expressed as means ⫾ SE.
Data were analyzed using SigmaStat (Systat Software). Unpaired
Student’s t-test was used to compare two different experimental
groups. One-way ANOVA followed by Holm-Sidak post hoc test was
used to compare more than two experimental groups. Results were
considered significant when P ⬍ 0.05.
RESULTS
Expression of D1-like receptors in gills of the blue crab.
Various in-house and commercially available antibodies against
D1-like dopamine receptors were tested on blue crab gill
homogenates. A rabbit polyclonal antibody generated against
the human D5R (GeneTex antibody) was used on samples from
HEK-293 cells stably expressing the human D5R (HEK-hD5R)
and on gills from the spiny lobster (P. interruptus) and the blue
crab (C. sapidus) (Fig. 1A). Three bands were visualized in
Fig. 1. Anti-dopamine D5 receptor (D5R) antibody (Ab) validation and D1R
expression in anterior and posterior gills. A: to validate its specificity, the
anti-D5R Ab (GeneTex) was used to visualize the human D5R in HEK-293
cells stably expressing the human D5R (HEK-hD5R), as well as the homolog
in the gill homogenates from the lobster (Panulirus interruptus) and blue crab
(Callinectes sapidus) in the absence or presence of its immunogen. An Ab
raised against the lobster anti-D1R [Clark et al. (14)] was also used to
visualize the D5R/D1R in all 4 homogenates, with or without the immunogen.
B: homogenates of anterior gills (AG) and posterior gills (PG) of C. sapidus
transferred to artificial seawater (10 ppt salinity) for 6 days were probed with
either the polyclonal anti-D5R Ab (D5R) alone or in the presence of 5 M excess
of its immunizing peptide. A major ⬃55-kDa band was visualized when
probed with the D5R Ab alone but not in the presence of the immunizing
peptide. The D1R was present in both AG and PG homogenates. Actin was
used as housekeeping protein. C: agarose gel analysis of RT-PCR products
from the gills of blue crab using degenerate primers. The amplicons were
resolved on a 1.5% agarose gel, stained with ethidium bromide, and visualized
using ultraviolet transillumination. Lane M: DNA size markers. Lane S:
RT-PCR products. Nucleotide sequence of the PCR amplicon (D) and pairwise
alignment of the amino acid sequence (E) derived from the PCR amplicon
(Can) with the corresponding region of the D1R of P. interruptus (Pan) and
Penaeus monodon (Pen).
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D1-LIKE DA RECEPTORS INHIBIT THE BLUE CRAB SODIUM PUMP
HEK-hD5R that correspond to glycosylated D5R (⬃125 kDa),
native D5R (⬃55 kDa), and presumably degradation products
(⬃30 –35 kDa). In the lobster homogenate, three bands were
visualized using the same antibody, i.e., ⬃125-kDa band, a
faint 55-kDa band, and ⬃45-kDa band, and two bands were
visualized in the blue crab homogenate, i.e., ⬃125- and 55-kDa
band. An antibody raised against the lobster anti-D1R (14)
was able to visualize ⬃55- and ⬃30-kDa bands in the HEKhD5R lysate, a single ⬃55-kDa band in the lobster homogenate, and ⬃120- and 55-kDa bands in the crab homogenate. All
of these bands disappeared when the corresponding immunogen was added into the antibody solution. It is conceivable that
the 55-kDa band in the crab homogenate, which was visualized
using both the GeneTex and the anti-lobster antibodies, represents the crab D1R, the homolog of the human D5R. We next
evaluated the expression of D1R in both anterior and posterior gills of the blue crab and found that it was expressed in
both structures (Fig. 1B). The ⬃55-kDa band was not visualized when the immunizing peptide was added. RT-PCR of the
cDNA prepared from the posterior gills amplified a predicted
⬃130-bp amplicon (Fig. 1C). Colony PCR and sequencing
revealed that the cloned amplicon contained 125 base pairs
(Fig. 1D). The amino acid sequence derived from the nucleotide sequence displayed 63 and 65% identity with corresponding regions of the D1R from P. interruptus and P. monodon,
respectively (Fig. 1E). These results suggest that the D1-like
receptor is expressed in the gills of the blue crab and shares
structural similarities with the arthropod and the rodent D1R
and the human D5R (6, 14).
The D1R expression in the posterior gills was not different
between crabs that were transferred to dilute seawater (10 ppt
salinity) and crabs in full-strength seawater (32 ppt salinity) for
6 days (96 ⫾ 6 vs. 100 ⫾ 5%, respectively; Fig. 2).
Localization of the D1-like receptor and Na⫹-K⫹-ATPase
␣-subunit in gills of the blue crab. Immunohistochemistry of
the posterior gills showed positive staining for both Na⫹-K⫹-
Fig. 2. Effect of salinity on the expression of D1R in posterior gills. Blue
crabs (C. sapidus) were transferred to artificial seawater with either 32 parts
per thousand (ppt) or 10 ppt salinity for 6 days. Gills 6 and 7 were excised and
the whole cell homogenate was prepared, resolved via 10% SDS-PAGE, and
probed for D1R and actin. D1-like dopamine receptor expression, normalized
to actin, was not significantly different (P ⬎ 0.05) between 32 ppt and 10 ppt,
Student’s t-test, n ⫽ 5/group. Immunoblots for each treatment are shown above
the corresponding bar graphs. Numerical data are expressed as means ⫾ SE.
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Fig. 3. Immunohistochemistry of the posterior gills. Posterior gill sections of
the blue crab C. sapidus in artificial seawater (10 ppt salinity) for 6 days were
incubated with anti-chicken Na⫹-K⫹-ATPase ␣-subunit (A) or anti-human
D5R dopamine receptor antibody (B and C), probed with donkey anti-mouse
secondary antibody, and counterstained with hematoxylin. Both Na⫹-K⫹ATPase ␣-subunit staining and D1R staining were more pronounced closer to
the central stem than towards the tip of the lamellae. Scale bar ⫽ 50 m.
ATPase ␣-subunit (Fig. 3A) and a D1R (Fig. 3, B and C) in
cells in the osmoregulatory patch along the central stem of the
posterior gills. Staining for both proteins was more evident
closer to the central stem compared with the outer edge of the
lamellae. The Na⫹-K⫹-ATPase ␣-subunit staining was primarily in the basolateral membrane (Fig. 4A), while the D1-like
dopamine receptor staining was in the basolateral membrane,
as well as the cytoplasm (Fig. 4C). The normal morphology of
the posterior gills is shown in the negative control images
Fig. 4. Immunohistochemistry of the posterior gills. Posterior gill sections of
the blue crabs C. sapidus transferred to artificial seawater (10 ppt salinity) for
6 days. A: sections incubated with anti-chicken Na⫹-K⫹-ATPase ␣-subunit.
B: negative control (no primary antibody). C: sections incubated with antihuman D5R antibody. D: negative control (no primary antibody). All sections
were probed with donkey anti-mouse secondary antibody and counterstained with hematoxylin. Both Na⫹-K⫹-ATPase ␣-subunit and D1R
were primarily localized in the basolateral membranes of the epithelial
cells. Scale bar ⫽ 50 m.
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D1-LIKE DA RECEPTORS INHIBIT THE BLUE CRAB SODIUM PUMP
Fig. 5. Colocalization of Na⫹-K⫹-ATPase ␣-subunit and D1R in posterior gills. Posterior gill sections of the blue crabs C. sapidus were immunostained for
Na⫹-K⫹-ATPase ␣-subunit (Alexa 488, green) and D1R (Alexa 568, red). Colocalization of both proteins is denoted by the yellow areas in the overlay image
(bottom left). Differential interference contrast (DIC) images were also obtained to show the boundaries (bottom right). Colocalization was observed both in cells
within the osmoregulatory patch and in cells near the tips of the lamellae. Scale bar ⫽ 50 um.
(Fig. 4, B and D). The cells found at the osmoregulatory
patch are larger than the other cells in the posterior gills.
These cells also have substantially more mitochondria and
possess most of the Na⫹-K⫹-ATPase activity of the lamellae (15).
Confocal microscopy revealed colocalization of the Na⫹⫹
K -ATPase ␣-subunit and the D1R at the basolateral
membrane as well as the cellular junctions and was observed
from the central stem to the tip of the lamellae (Fig. 5).
Effect of D1-like receptor stimulation on D1R and cAMP
levels. Posterior gills of crabs infused with fenoldopam (15
min) showed a slight decrease in D1R (55 kDa) compared
with control (100 ⫾ 1 vs. 87 ⫾ 2%; P ⬍ 0.05; Fig. 6). Infusion
with fenoldopam significantly increased cAMP production,
relative to vehicle infusion (42.2 ⫾ 2.5, n ⫽ 4, vs. 21.0 ⫾ 3.2
pmol cAMP/mg protein). To show that the effect was due to
D1-like dopamine receptors, crabs were infused first with the
D1-like receptor antagonist SCH23390 for 15 min, followed by
fenoldopam. SCH23390 abolished the stimulatory effect of
fenoldopam (21.2 ⫾ 5.5 pmol cAMP/mg protein). SCH23390
did not have a significant effect when infused alone (19.5 ⫾ 1.9
pmol cAMP/mg protein; Fig. 7).
Na⫹-K⫹-ATPase activity in response to D1-like receptor
stimulation. Posterior gills of crabs infused with fenoldopam
showed a significant decrease in Na⫹-K⫹-ATPase activity
compared with vehicle infusion [fenoldopam ⫽ 13.3 ⫾ 0.9
Pi·mg protein⫺1·h⫺1 vs. vehicle (control) ⫽ 24.6 ⫾ 1.9 mol
Pi·mg protein⫺1·h⫺1; Fig. 8]. To show that the decrease in
activity was due to D1-like dopamine receptors, the D1-like
receptor antagonist SCH23390 was infused alone or with
fenoldopam. The infusion of SCH23390 alone did not affect
the activity of Na⫹-K⫹-ATPase (SCH23390 ⫽ 22.8 ⫾ 1.2
mol Pi·mg protein⫺1·h⫺1) but abolished the decrease in the
activity elicited by fenoldopam infusion (SCH23390 ⫹
fenoldopam ⫽ 23.4 ⫾ 2.0 mol Pi·mg protein⫺1·h⫺1; Fig. 8).
There were no differences in the ouabain-insensitive ATPase
activity among the groups, indicating that the activity of
ATPases other than Na⫹-K⫹-ATPase was similar in all groups
(data not shown). Fenoldopam infusion did not produce any
change in the expression of the Na⫹-K⫹-ATPase ␣-subunit
(Fig. 9), indicating that the decrease in Na⫹-K⫹-ATPase activity was not related to the decreased expression of the
protein.
Fig. 6. Effect of fenoldopam on the expression of D1R. Blue crabs
(C. sapidus) in artificial seawater (10 ppt salinity) for 6 days were infused with
vehicle plus fenoldopam or with vehicle alone (control). Posterior gills 6 and
7 were excised, and whole gill tissue homogenate was prepared, resolved via
10% SDS-PAGE, and probed with polyclonal D5R antibody and with goat
anti-rabbit secondary antibody conjugated with horseradish peroxidase. Bands
were visualized using chemiluminescence. Actin was used as housekeeping
protein. Numerical data are expressed as means ⫾ SE. Fenoldopam-infused
crabs showed a small, but significant, decrease in expression compared with
control (0.81 ⫾ 0.02 vs. 0.74 ⫾ 0.02, n ⫽ 5/treatment). *P ⬍ 0.05 vs. control,
Student’s t-test. Immunoblots for each treatment are shown above the corresponding bar graphs.
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D1-LIKE DA RECEPTORS INHIBIT THE BLUE CRAB SODIUM PUMP
Fig. 7. cAMP levels in gills in response to vehicle or drug infusion. Blue crabs
(C. sapidus) transferred to artificial seawater (10 ppt salinity) for 6 days were
infused with vehicle plus the indicated drug or with vehicle alone (control).
Posterior gills 6 and 7 were excised, and the whole gill homogenates were
assayed for cAMP (normalized for total protein concentration). Numerical data
are expressed as means ⫾ SE. Fenoldopam (Fen), a D1R/D5R agonist,
significantly increased cAMP levels relative to control (Con). SCH23390
(SCH), a D1R/D5R antagonist, which by itself had no effect, blocked the
stimulatory effect of fenoldopam (SCH ⫹ Fen) on cAMP levels. *P ⬍ 0.05 vs.
all other treatments, one-way ANOVA followed by Holm-Sidak posttest, n ⫽
3/treatment.
DISCUSSION
This study reports the presence of a D1-like dopamine
receptor, which is most likely the D1R, in the posterior gills
of the Atlantic blue crab C. sapidus. When D1-like dopamine
receptors were stimulated with the D1-like receptor agonist
fenoldopam in intact crabs, the posterior gills responded with
an increase in cAMP production and a decrease in Na⫹-K⫹ATPase activity without a change in expression of its ␣-subunit. Previous studies have indicated the presence of a dopamine receptor in crustaceans (14, 21, 23, 37, 46). However,
among crustaceans, it is only in the spiny lobster P. interruptus
that two D1-like receptors, the D1␣R and D1R, have been
described so far (14). In this study, an ⬃55-kDa band was
visualized in the gills, which conceivably corresponds to the
crab D1R. RT-PCR on cDNA prepared from the posterior
gills also indicated the presence of D1R transcript.
Compared with previous studies, the present study employed
two novel techniques to study the physiological response of the
Fig. 8. Na⫹-K⫹-ATPase activity in gills in response to drug infusion. Blue
crabs (C. sapidus) transferred to artificial seawater (10 ppt salinity) for 6 days
were infused with vehicle plus indicated drug or with vehicle alone (control).
Posterior gills 6 and 7 were excised and the whole gill homogenates were
assayed for Na⫹-K⫹-ATPase activity (normalized for the total protein concentration). Numerical data are expressed as means ⫾ SE. Fenoldopam, a
D1R/D5R agonist, significantly decreased Na⫹-K⫹-ATPase activity relative to
control. SCH23390, a D1R/D5R antagonist that by itself had no effect, blocked
the effect of fenoldopam on Na⫹-K⫹-ATPase activity. For abbreviations, see
Figure 7. *P ⬍ 0.05 vs. other treatments, one-way ANOVA followed by
Holm-Sidak posttest, n ⫽ 8 –12/group.
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Fig. 9. Effect of fenoldopam on the expression of Na⫹-K⫹-ATPase ␣-subunit
in gills. Blue crabs (C. sapidus) transferred to artificial seawater (10 ppt
salinity) for 6 days were infused with vehicle plus fenoldopam, or with vehicle
alone (control). Posterior gills 6 and 7 were excised, and whole gill tissue
homogenates were prepared, resolved via 10% SDS-PAGE, and probed for
Na⫹-K⫹-ATPase and actin (used as a housekeeping protein). Fenoldopaminfused crabs showed no change in Na⫹-K⫹-ATPase expression (n ⫽ 4)
compared with control (n ⫽ 3).
dopamine receptor in the posterior gills of blue crabs. First, the
experiments in the current study were performed on living
crabs and not on isolated gills. It is difficult to examine the in
vivo effects of perfused drugs in isolated gills of C. sapidus
since properly securing a closed circuit system necessitates
clamping of the base of the gill where a large portion of the ion
transporting cells are located (1, 32). Second this study used a
continuous drug infusion technique that allows for continuous
bioavailability of the drug and a more stable pharmacokinetic
profile (17, 49). In contrast, previous studies have employed
the technique of administering a single bolus of a pharmacological agent into the crabs (37, 44, 45).
The dopamine receptor and the sodium pump colocalized
along the basolateral membrane, especially in cells within the
osmoregulatory patch. In addition to showing a potential for
the interaction between the sodium pump and the dopamine
receptor, these results suggest that the dopamine receptor may
also play an important role in the regulation of sodium uptake
in the posterior gills that may be associated with the osmoregulatory response. Acute treatment with the D1-like receptor
agonist fenoldopam resulted in a slight decrease in D1R
expression, which may be an acute compensatory mechanism
to osmoregulate in lower salinities. In addition, the decrease in
expression may indicate receptor degradation, similar to what
occurs in the mammalian system (31).
Previous studies in crustaceans have shown that the D1␣R
(D1R in mammals) is coupled to the stimulatory G protein Gs␣,
while the D1R (D5R in mammals) is coupled to stimulatory
Gs␣ and Gq␣, both of which increase cAMP production (14).
In contrast, the D2␣PanR, a D2-like dopamine receptor, is
coupled to inhibitory G␣i/␣o, which decreases cAMP production (13, 14). Thus changes in the level of intracellular cAMP
can be used to monitor the physiological response of the
dopamine receptors upon agonist activation. In the current
study, the stimulation of the D1-like receptors using fenoldopam led to an increase in cAMP production, indicating that the
D1-like receptor was functional and that its signal transduction
pathway was intact in the posterior gills.
Treatment with fenoldopam led to a decrease in Na⫹-K⫹ATPase activity without a concomitant change in the expression of the Na⫹-K⫹-ATPase-␣-subunit expression. The Na⫹K⫹-ATPase expression data suggest that the decrease in activity was not due to the degradation of the transporter but rather
to an alteration of the intrinsic conformation of the sodium
pump, resulting in its internalization and inactivation. It is
conceivable that the activation of Na⫹-K⫹-ATPase observed in
previous studies using high concentrations of dopamine,
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D1-LIKE DA RECEPTORS INHIBIT THE BLUE CRAB SODIUM PUMP
fenoldopam, and SCH23390 is not due to the activation of the
D1-like dopamine receptors alone but to the concomitant activation of other receptors, e.g., serotonin receptors (vide infra).
The role of D2-like receptors in the osmoregulation of euryhaline crabs should be also considered because it has been shown
that the perfusion of their posterior gills with spiperone and
domperidone, two potent D2-like dopamine receptor antagonists, blocked the ability of dopamine to increase the transepithelial potential difference (21). Previous studies have mostly
used the natural ligand for the dopamine receptors, dopamine,
as agonist; hence, the activation of the D2-like dopamine
receptors is likely to occur in this context since dopamine is
able to activate G␣s, G␣q, and G␣i in crustaceans (13, 14).
Under certain circumstances, the mammalian D3R, a D2-like
dopamine receptor, can be linked to G␣s (40). Thus it is
possible that an increase or a decrease in cAMP can ensue after
dopamine activation, depending on which cascade dominates
the response. However, what regulates the eventual response
must be determined.
Previous studies have shown that an increase in cAMP
production in the osmoregulatory gills of euryhaline crustaceans is associated with an increase in Na⫹-K⫹-ATPase activity and a transient activation followed by a subsequent inhibition of transepithelial potential difference (21, 23, 44). These
authors suggested that the transient increase in cAMP content
was responsible for Na⫹-K⫹-ATPase stimulation. However,
direct proof of this hypothesis has not been forthcoming. It
would be interesting to determine whether D1- and D2-like
receptors interact in the regulation of Na⫹-K⫹-ATPase in
crustacean gills. This occurs in mammalian renal tubule cells
under conditions of moderate extracellular fluid expansion,
where the D2-like receptors act synergistically with the D1-like
receptors to increase sodium excretion by inhibiting Na⫹-K⫹ATPase activity (3, 16, 29).
In mammals, higher concentrations of dopamine can activate
nondopamine receptors, e.g., serotonin and adrenergic receptors. There are studies showing that D1-like receptor drugs,
such as fenoldopam and SCH23390, have high affinities for
serotonin receptors (12, 35), a possibility with the high concentrations (10 M) used in previous reports. Several crustacean serotonin receptors, which also increase cAMP production upon stimulation, have been reported in the somatogastric
ganglion but not in the gills; these crustacean serotonin receptors are not pharmacologically similar to vertebrate serotonin
receptors (14, 46, 47). Octopamine (OA), a biogenic monoamine structurally related to norepinephrine, acts as a neurohormone, neuromodulator, and neurotransmitter in invertebrates, including the crustaceans. Activation of the OA receptors, which are similar to the mammalian adrenergic receptors,
also invariably results in increased cAMP levels (42). These
receptors, if proven to be present in the posterior gills, could
conceivably account for the previous observations subsequent
to the treatment with dopamine.
Perspectives and Significance
In summary, we have provided evidence for the presence of
dopamine receptors, presumably the D1R, which colocalize
with the Na⫹-K⫹-ATPase at the basolateral membrane of the
cells that populate the osmoregulatory patch of the posterior
gills of the blue crab. While we have shown that agonist
stimulation of these receptors resulted in diminished receptor
abundance, increased cAMP production, and decreased sodium
transport via the inhibition of Na⫹-K⫹-ATPase activity, the
details of the molecular mechanisms involved in this process
have yet to be established. The inhibition of Na⫹-K⫹-ATPase
activity by the D1-like dopamine receptors appears to have
been conserved in phylogenetically distinct organisms. In
mammalian renal tubule cells under conditions of moderate
sodium excess, the D1-like dopamine receptors are responsible
for decreasing renal sodium transport by at least 50% (10, 18,
24, 50, 51). Mammalian renal tubule cells can adapt in an
environment of low (50 mosmol/kgH2O) to high osmolality
(1,200 mosmol/kgH2O) (30), similar to conditions to which
euryhaline crustaceans are exposed during their migration from
low- to high-salinity water. TonEBP (30), which plays an
important role in the renal tubular response to hypertonicity,
stimulates the renal proximal tubule production of dopamine
(27). A blueprint for the regulation of sodium transport in
specialized tissues is shared by various organisms belonging to
various phyla; however, unique variations in terms of novel
interacting proteins or novel functions for the same protein
exist to allow the organisms to thrive in their own biological
niche. It would be interesting to determine if the D1- and
D2-like receptors interact in the regulation of Na⫹-K⫹-ATPase
in crustacean gills and establish the cross talk between dopamine and other hormones in osmoregulation. Understanding
the underlying mechanisms may shed light on the evolution of
the pathways involved in ionic and osmotic regulation.
ACKNOWLEDGMENTS
We thank Dr. Deborah J. Baro, Georgia State University, for the rabbit
anti-lobster dopamine receptor antibodies and immunizing peptides that were
used to verify the crab D1R.
GRANTS
This work was supported in part by National Institutes of Health Grants
R01-HL-092196, R37-HL-023081, R01-DK-090918, and R01-DK-039308.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: F.B.A., V.A.M.V., I.A., P.A.J., and G.P.C. conception and design of research; F.B.A., V.A.M.V., P.R.K., S.A.O., L.D.A., J.E.J.,
and J.Y. performed experiments; F.B.A., V.A.M.V., P.R.K., S.A.O., L.D.A.,
D.L.L., I.A., P.A.J., and G.P.C. analyzed data; F.B.A., V.A.M.V., P.R.K.,
L.D.A., J.Y., D.L.L., I.A., P.A.J., and G.P.C. interpreted results of experiments; V.A.M.V., P.R.K., and P.A.J. prepared figures; V.A.M.V., J.E.J., J.Y.,
D.L.L., P.A.J., and G.P.C. edited and revised manuscript; V.A.M.V., P.R.K.,
L.D.A., J.E.J., J.Y., D.L.L., I.A., P.A.J., and G.P.C. approved final version of
manuscript; I.A. drafted manuscript.
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