D1-like dopamine receptors downregulate Na�-K�-ATPase activityand increase cAMP production in the posterior gills of the blue crabCallinectes 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
1The 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 ofMedicine, University of Maryland School of Medicine, Baltimore, Maryland; 4Department of Biology, The College of NewJersey, 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, AsicoLD, 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 poste-rior gills of the blue crab Callinectes sapidus. Am J Physiol RegulIntegr Comp Physiol 307: R634 –R642, 2014. First published July30, 2014; doi:10.1152/ajpregu.00555.2013.—Dopamine-mediatedregulation of Na�-K�-ATPase activity in the posterior gills of somecrustaceans has been reported to be involved in osmoregulation. Thedopamine receptors of invertebrates are classified into three groupsbased on their structure and pharmacology: D1- and D2-like receptorsand a distinct invertebrate receptor subtype (INDR). We tested thehypothesis that a D1-like receptor is expressed in the blue crabCallinectes sapidus and regulates Na�-K�-ATPase activity. RT-PCR,using degenerate primers, showed the presence of D1�R mRNA in theposterior gill. The blue crab posterior gills showed positive immuno-staining for a dopamine D5 receptor (D5R or D1�R) antibody in thebasolateral membrane and cytoplasm. Confocal microscopy showedcolocalization of Na�-K�-ATPase and D1�R in the basolateral mem-brane. To determine the effect of D1-like receptor stimulation onNa�-K�-ATPase activity, intact crabs acclimated to low salinity for 6days were given an intracardiac infusion of the D1-like receptoragonist fenoldopam, with or without the D1-like receptor antagonistSCH23390. Fenoldopam increased cAMP production twofold anddecreased Na�-K�-ATPase activity by 50% in the posterior gills.This effect was blocked by coinfusion with SCH23390, which had noeffect on Na�-K�-ATPase activity by itself. Fenoldopam minimallydecreased D1�R protein expression (10%) but did not affect Na�-K�-ATPase �-subunit protein expression. This study shows the pres-ence of functional D1�R in the posterior gills of euryhaline crabschronically exposed to low salinity and highlights the evolutionarilyconserved function of the dopamine receptors on sodium homeostasis.
dopamine receptor; Na�-K�-ATPase; blue crab; cAMP; posteriorgills
THE ATLANTIC BLUE CRAB, Callinectes sapidus, a euryhalinecrustacean, must osmoregulate to survive in rapidly changingsaline environments of estuarine habitats. It is considered to bea strong hyperosmoregulator that can maintain an almost con-stant 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, isone of several osmoregulatory mechanisms in euryhaline crus-taceans (25, 33, 39, 43, 44). However, the regulatory pathwaysleading to modulation of Na�-K�-ATPase activity in posteriorgills of euryhaline crustaceans are not fully understood.
Dopamine has been shown to increase the Na�-K�-ATPaseactivity in posterior gills of crustaceans through a cAMP-dependent pathway (36). Invertebrates have three subfamiliesof dopamine receptors, i.e., 1) the DOP1 subfamily, which isrelated to vertebrate D1-like receptors; 2) the INDR subfamily,which is a distinct invertebrate group that functionally behaveslike vertebrate D1-like receptors; and 3) the invertebrate D2-like receptor subfamily, which is related to vertebrate D2-likereceptors (38). Specifically in crustaceans, there are two typesof D1-like receptors, D1�PAN and D1�PAN (also termed D1Rand D5R in humans, respectively). D1-like receptors stimulate,while D2-like receptors inhibit, adenylyl cyclase activity invertebrates and invertebrates (6, 14). D1-like receptors havebeen pharmacologically characterized in the crustacean Erio-cheir sinensis (36); however, the role of this receptor subtypein the regulation of Na�-K�-ATPase in response to changes inenvironmental osmolality is still unclear.
In mammals, there are currently two paradigms of theD1-like dopamine receptor effect on ion transport that act inopposite manner, depending on the cell type. In human lungepithelia, dopamine via D1-like dopamine receptors increasessodium transport by stimulating the rapid recruitment of Na�-K�-ATPase from cellular endosomes to the basolateral mem-brane (5). In the proximal and distal tubules of the mammaliankidney, however, dopamine decreases ion transport by actingon D1-like dopamine receptors to increase cAMP, which leadsto the phosphorylation of Na�-K�-ATPase, resulting in itsinternalization and inactivation (2, 4, 9, 11, 22, 26). Alteredarachidonic metabolism may result in the failure of dopamineto inhibit Na�-K�-ATPase (28). The objective of the currentstudy was to test the hypothesis that D1-like receptors areexpressed in the posterior gills of the euryhaline blue crab C.sapidus and function to increase cAMP production to ulti-mately regulate Na�-K�-ATPase activity.
MATERIALS AND METHODS
Animals. Male blue crabs in intermolt were collected from theAnnapolis area and Hoopers Island, Chesapeake Bay, MD, betweenJune-October and housed at 25°C in filtered recirculating tanks
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:[email protected]).
Am J Physiol Regul Integr Comp Physiol 307: R634–R642, 2014.First published July 30, 2014; doi:10.1152/ajpregu.00555.2013.
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 widthsfrom 11 to 15 cm. The crabs were fed once daily with a diet consistingof processed oysters and dried pellet food. The crabs were exposed toa 12:12-h light-dark photoperiod and, after exposure to dilute seawaterfor 6 days, examined before experimentation. This duration of expo-sure was adequate to stimulate the hypoosmotic response in the crabsand to upregulate expression of Na�-K�-ATPase in the epithelialcells of the gills (33).
Drug infusion. Crabs undergoing drug infusion were removed fromthe aerated tanks containing 10 ppt artificial seawater on day 5 ofacclimation (32, 33), and a 2-mm hole was drilled through thecarapace directly above the heart cavity, as described by Burnett et al.(7). The drill-bit was pressed onto the carapace to create a depressiondeep enough to allow needle-stick penetration but not cause anybleeding. Latex rubber and cyanoacrylate adhesive were used to coverthe depression to prevent any hemolymph bleed out caused by thepuncture. The crabs were allowed to recover for 24 h before the study.Subsequently, vehicle (137 mM NaCl, 3 mM KCl, 5 mM MgSO4, and3 mM HEPES, pH 7.4) that is isosmotic with the crab’s hemolymph,with or without drugs (1 �M fenoldopam and 5 �M SCH23390), wasinfused directly (0.1 ml/min for 15 min) into the heart via an 18-gaugeneedle connected to an infusion pump. Initial experiments usinglissamine green directly infused into the heart showed that the gillswere fully perfused within 5 min. The fenoldopam (1 �M) andSCH23390 (5 �M) doses in our studies were based on studies in ratsin which the drugs were infused directly into the renal artery (18, 53).These doses were lower than those used in the shore crab Chasmag-nathus granulatus to avoid targeting other receptors, e.g., serotoninreceptors (12, 35), which may occur when higher doses are used. Theperfusion rate of 0.1 ml/min used was the same infusion rate used toperfuse the gills of C. granulatus (21). A drug infusion period of 15min was chosen because the D1-like receptor was phosphorylated andinternalized into the cell cytoplasm following 15 min of exposure todopamine in human embryonic kidney cells heterologously express-ing the rat D1R (41).
Whole gill homogenate. The crabs were anesthetized by being putin ice for 20 min and then killed by carapace removal. Gills 6 and 7were excised to represent the posterior gills while gills 3 and 4 wereexcised to represent the anterior gills. The gills were blotted dry andhomogenized in ice-cold buffer (250 mM sucrose, 2 mM EDTA, and50 mM imidazole, pH 7.2) for Na�-K�-ATPase activity and cAMPproduction assays or lysis buffer (1% Triton X, 0.1% SDS, and 0.5%sodium deoxycholate) for immunoblotting studies. A protease inhib-itor cocktail (10 mM AEBSF, 1 mM trypsin, and 10 mM PMSF) wasadded to prevent proteolysis. Saponin (20 �g/mg protein) was used topermeabilize the membranes to maximize substrate accessibility forthe endogenous Na�-K�-ATPase. The crude homogenates were par-tially purified by centrifuging at 10,000 g. The final protein concen-tration (BCA kit; Pierce, Rockford, IL) of each supernatant wasadjusted to 1.0–1.5 mg/ml before storage at �80°C for subsequentstudies.
Immunoblotting. Samples of uniform amounts of protein wereresolved via 10% SDS-PAGE (Invitrogen, Carlsbad, CA). Proteinswere electrotransferred onto nitrocellulose membranes (Bio-Rad, Her-cules, CA) using a wet transfer apparatus (Invitrogen) and subjectedto immunoblotting, as reported previously (19, 52, 54). The primaryantibodies used were rabbit polyclonal anti-human D5R (Genetex, SanAntonio, TX), mouse monoclonal anti-chicken Na�-K�-ATPase�-subunit (developed by D. M. Fambrough and obtained from theDevelopmental Studies Hybridoma Bank, The University of Iowa,Department of Biological Sciences, Iowa City, IA), and mouse nanti-actin (Sigma-Aldrich, St. Louis, MO). The rabbit anti-lobsterD1�R antibody and immunogen were kindly provided by Dr. DeborahJ. Baro, Georgia State University. Donkey anti-mouse and goatanti-rabbit secondary antibodies (Santa Cruz Biotechnology, Santa
Cruz, CA) were used. The mouse monoclonal anti-chicken Na�-K�-ATPase �-subunit antibody has been used successfully for a range ofarthropods, including C. sapidus (34, 48). The mouse monoclonalanti-actin antibody is reactive to many species, including Drosophila.The published immunogen for actin shares 71% sequence identitywith that of Drosophila and 79% with that of Callinectis.
The bands were visualized using an enhanced chemiluminescencedetection kit (Millipore, Billerica, MA) or by IR Western blot detec-tion via the Odyssey Imager (Li-COR, Lincoln, NE) and quantified bydensitometric scan (Scion Images, Frederick, MD). Actin was used asthe housekeeping protein. To determine the D5R epitope specificity,the immunoblots were incubated in antibody solution preincubatedwith or without the blocking peptide (cat. no. GTX77969; Genetex,San Antonio, TX). The company has not disclosed the immunogensequence but revealed that a pairwise alignment of the sequence of theimmunogen and that of Panulirus interruptus D1�R (the closestknown homolog) showed 28% sequence identity. Total cell lysatesfrom HEK-293 cells heterologously expressing the human D5R andtotal gill homogenates from the spiny lobster P. interruptus that weresupplemented with a protease inhibitor cocktail were used as positivecontrols.
Degenerate primers and RT-PCR. Forward degenerate primerswere designed using the amino acid sequences YIHIKD (forwardprimer: 5=-tayathcayathaargay-3=) of the D1-like receptors from theseven arthropods phylogenetically closest to the blue crab. Of thesespecies, 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 pi-sum (XP_001947683), P. interruptus (DQ295791), and Penaeusmonodon (JQ901712) showed that the sequence YIHIKD is highlyconserved in the D5R of these seven species. Four reverse primers of20–23 basepairs (23-bp reverse primer; 5=-GGGGTTTGTGGAGCT-TGAGGCTG-3=) varying in their sizes at the 3=-end were derivedfrom the 100-bp nucleotide sequence of the D1�R of the Celucapugilator (http://www.genome.ou.edu/FiddlerCrab_Illumina_seqs/A4_B1_498828_100bp). RNA was isolated from the crab posteriorgills using the RNeasy mini kit (Qiagen, Valencia, CA), reversetranscribed and amplified via PCR using four reverse primers mixedin equimolar concentration (1 �M) in combination with the forwardprimer (1 �M). The PCR products were resolved in 2% agarose geland visualized under ultraviolet light after staining with ethidiumbromide. The PCR amplicon was purified from the agarose gel andcloned in to pCR4-TOPO cloning vector for further colony PCR andsequence analyses.
Immunostaining and confocal microscopy. Crabs were acclimatedto 32 ppt salinity for 6 days before death and excision of gills 6 and7. The gills were perfused with PBS then washed and stored in thehistological fixative HistoChoice (AMRESCO, Solon, OH) for 3 daysat 4°C. Four-millimeter-thick sections of the posterior gills wereprepared for microscopy. For immunohistochemistry, the sectionswere incubated with either anti-D5R antibody or anti-Na�-K�-ATPase�-subunit antibody for 2 h at room temperature. After being washedwith PBS, the sections were incubated with donkey anti-mouse andgoat anti-rabbit secondary antibodies for 1 h at room temperature. Thebiotinylated secondary antibodies were visualized using the ABCcomplex kit (Pierce) and Vector VIP (Vector Laboratories, Burlin-game, CA), counterstained with hematoxylin (Sigma-Aldrich), andviewed with a Nikon E600 digital microscope.
For confocal microscopy, the slides were double immunostainedwith rabbit anti-D5R antibody and mouse anti-Na�-K�-ATPase�-subunit antibody for 2 h at room temperature and probed with goatanti-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 anddistilled water once, the coverslips were mounted on glass slides usingVectashield mounting medium and sealed with nail polish. Negative
R635D1-LIKE DA RECEPTORS INHIBIT THE BLUE CRAB SODIUM PUMP
controls were likewise prepared but with the omission of the primaryantibodies. Colocalization of D1�R and the Na�-K�-ATPase �-sub-unit was evaluated by laser scanning confocal microscopy, using anOlympus Fluoview FV300 inverted microscope using 450-nm exci-tation and 535-nm emission filters for Alexa Fluor 488- and 560-nmexcitation and 645-nm emission filters for Alexa Fluor 568. Imageswere overlaid using Olympus Fluoview FV300 version 3C Acquisi-tion Software to determine colocalization.
cAMP assay. Whole gill homogenates were pipetted into platewells containing cAMP-specific rabbit antibody binding sites andcompetitive cAMP-acetylcholinesterase (AChe) conjugate (CaymanChemical, Ann Arbor, MI). The plate was incubated overnight at 4°Cafter which the wells were washed. The plate was developed withEllman’s reagent, which contains the AChe substrate, and incubated
in the dark for 2 h at room temperature. cAMP was quantified (412nm) using a spectrophotometer (Thermofisher Scientific). cAMP con-tent of the samples was calculated compared with known standardsand expressed as picomoles of cAMP per milligram of protein.
Measurement of Na�-K�-ATPase activity. Na�-K�-ATPase activ-ity was measured (26) in an incubation buffer containing 140 mMNaCl, 5 mM KCl, 5 mM MgCl2, 30 mM Tris·HCl, 1 mM EGTA, 3mM Na2ATP, and [�-32P]ATP (2–5 Ci/mmol) in tracer amounts (5nCi/�l), using a 15-min incubation period at 37°C in the presence orabsence of 2 mM ouabain, a Na�-K�-ATPase inhibitor. Whenouabain was present, NaCl and KCl were omitted from the incubationbuffer. The reaction was initiated by the addition of 10 �l of homog-enate to 90 �l of incubation buffer. The reaction was terminated bythe addition of activated charcoal/trichloroacetic acid and rapid cool-ing on ice. After 1 h, the samples were centrifuged for 4 min at 14,000rpm to separate the charcoal that contained the unhydrolyzed nucle-otide. Radioactivity present in the supernatant containing the inor-ganic [�-32P] produced by ATPase activity was measured in quadru-plicate using a liquid scintillation spectrometer. Na�-K�-ATPaseactivity was calculated by subtracting the ouabain-insensitive ATPaseactivity from the total ATPase activity and expressed as millimoles ofPi per milligram of protein per hour.
Chemicals. Fenoldopam bromide and SCH23390 were purchasedfrom Sigma-Aldrich. [�-32P]ATP was purchased from Perkin-ElmerLife Sciences. All other chemicals were obtained from Sigma-Al-drich.
Statistical analysis. Numerical data are expressed as means � SE.Data were analyzed using SigmaStat (Systat Software). UnpairedStudent’s t-test was used to compare two different experimentalgroups. One-way ANOVA followed by Holm-Sidak post hoc test wasused to compare more than two experimental groups. Results wereconsidered significant when P 0.05.
RESULTS
Expression of D1-like receptors in gills of the blue crab.Various in-house and commercially available antibodies againstD1-like dopamine receptors were tested on blue crab gillhomogenates. A rabbit polyclonal antibody generated againstthe human D5R (GeneTex antibody) was used on samples fromHEK-293 cells stably expressing the human D5R (HEK-hD5R)and on gills from the spiny lobster (P. interruptus) and the bluecrab (C. sapidus) (Fig. 1A). Three bands were visualized in
Fig. 1. Anti-dopamine D5 receptor (D5R) antibody (Ab) validation and D1�Rexpression in anterior and posterior gills. A: to validate its specificity, theanti-D5R Ab (GeneTex) was used to visualize the human D5R in HEK-293cells stably expressing the human D5R (HEK-hD5R), as well as the homologin the gill homogenates from the lobster (Panulirus interruptus) and blue crab(Callinectes sapidus) in the absence or presence of its immunogen. An Abraised against the lobster anti-D1�R [Clark et al. (14)] was also used tovisualize the D5R/D1�R in all 4 homogenates, with or without the immunogen.B: homogenates of anterior gills (AG) and posterior gills (PG) of C. sapidustransferred to artificial seawater (10 ppt salinity) for 6 days were probed witheither the polyclonal anti-D5R Ab (D5R) alone or in the presence of 5 M excessof its immunizing peptide. A major 55-kDa band was visualized whenprobed with the D5R Ab alone but not in the presence of the immunizingpeptide. The D1�R was present in both AG and PG homogenates. Actin wasused as housekeeping protein. C: agarose gel analysis of RT-PCR productsfrom the gills of blue crab using degenerate primers. The amplicons wereresolved on a 1.5% agarose gel, stained with ethidium bromide, and visualizedusing ultraviolet transillumination. Lane M: DNA size markers. Lane S:RT-PCR products. Nucleotide sequence of the PCR amplicon (D) and pairwisealignment of the amino acid sequence (E) derived from the PCR amplicon(Can) with the corresponding region of the D1�R of P. interruptus (Pan) andPenaeus monodon (Pen).
R636 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 werevisualized using the same antibody, i.e., 125-kDa band, afaint 55-kDa band, and 45-kDa band, and two bands werevisualized in the blue crab homogenate, i.e., 125- and 55-kDaband. An antibody raised against the lobster anti-D1�R (14)was able to visualize 55- and 30-kDa bands in the HEK-hD5R lysate, a single 55-kDa band in the lobster homoge-nate, and 120- and 55-kDa bands in the crab homogenate. Allof these bands disappeared when the corresponding immuno-gen was added into the antibody solution. It is conceivable thatthe 55-kDa band in the crab homogenate, which was visualizedusing both the GeneTex and the anti-lobster antibodies, repre-sents the crab D1�R, the homolog of the human D5R. We nextevaluated the expression of D1�R in both anterior and poste-rior gills of the blue crab and found that it was expressed inboth structures (Fig. 1B). The 55-kDa band was not visual-ized when the immunizing peptide was added. RT-PCR of thecDNA prepared from the posterior gills amplified a predicted130-bp amplicon (Fig. 1C). Colony PCR and sequencingrevealed that the cloned amplicon contained 125 base pairs(Fig. 1D). The amino acid sequence derived from the nucleo-tide sequence displayed 63 and 65% identity with correspond-ing regions of the D1�R from P. interruptus and P. monodon,respectively (Fig. 1E). These results suggest that the D1-likereceptor is expressed in the gills of the blue crab and sharesstructural similarities with the arthropod and the rodent D1�Rand the human D5R (6, 14).
The D1�R expression in the posterior gills was not differentbetween crabs that were transferred to dilute seawater (10 pptsalinity) and crabs in full-strength seawater (32 ppt salinity) for6 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 ofthe posterior gills showed positive staining for both Na�-K�-
ATPase �-subunit (Fig. 3A) and a D1�R (Fig. 3, B and C) incells in the osmoregulatory patch along the central stem of theposterior gills. Staining for both proteins was more evidentcloser to the central stem compared with the outer edge of thelamellae. The Na�-K�-ATPase �-subunit staining was primar-ily in the basolateral membrane (Fig. 4A), while the D1-likedopamine receptor staining was in the basolateral membrane,as well as the cytoplasm (Fig. 4C). The normal morphology ofthe posterior gills is shown in the negative control images
Fig. 2. Effect of salinity on the expression of D1�R in posterior gills. Bluecrabs (C. sapidus) were transferred to artificial seawater with either 32 partsper thousand (ppt) or 10 ppt salinity for 6 days. Gills 6 and 7 were excised andthe whole cell homogenate was prepared, resolved via 10% SDS-PAGE, andprobed for D1�R and actin. D1-like dopamine receptor expression, normalizedto 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 abovethe corresponding bar graphs. Numerical data are expressed as means � SE.
Fig. 3. Immunohistochemistry of the posterior gills. Posterior gill sections ofthe blue crab C. sapidus in artificial seawater (10 ppt salinity) for 6 days wereincubated with anti-chicken Na�-K�-ATPase �-subunit (A) or anti-humanD5R dopamine receptor antibody (B and C), probed with donkey anti-mousesecondary antibody, and counterstained with hematoxylin. Both Na�-K�-ATPase �-subunit staining and D1�R staining were more pronounced closer tothe central stem than towards the tip of the lamellae. Scale bar � 50 �m.
Fig. 4. Immunohistochemistry of the posterior gills. Posterior gill sections ofthe blue crabs C. sapidus transferred to artificial seawater (10 ppt salinity) for6 days. A: sections incubated with anti-chicken Na�-K�-ATPase �-subunit.B: negative control (no primary antibody). C: sections incubated with anti-human D5R antibody. D: negative control (no primary antibody). All sectionswere probed with donkey anti-mouse secondary antibody and counter-stained with hematoxylin. Both Na�-K�-ATPase �-subunit and D1�Rwere primarily localized in the basolateral membranes of the epithelialcells. Scale bar � 50 �m.
R637D1-LIKE DA RECEPTORS INHIBIT THE BLUE CRAB SODIUM PUMP
(Fig. 4, B and D). The cells found at the osmoregulatorypatch are larger than the other cells in the posterior gills.These cells also have substantially more mitochondria andpossess most of the Na�-K�-ATPase activity of the lamel-lae (15).
Confocal microscopy revealed colocalization of the Na�-K�-ATPase �-subunit and the D1�R at the basolateralmembrane as well as the cellular junctions and was observedfrom the central stem to the tip of the lamellae (Fig. 5).
Effect of D1-like receptor stimulation on D1�R and cAMPlevels. Posterior gills of crabs infused with fenoldopam (15min) showed a slight decrease in D1�R (55 kDa) comparedwith control (100 � 1 vs. 87 � 2%; P 0.05; Fig. 6). Infusionwith fenoldopam significantly increased cAMP production,relative to vehicle infusion (42.2 � 2.5, n � 4, vs. 21.0 � 3.2pmol cAMP/mg protein). To show that the effect was due toD1-like dopamine receptors, crabs were infused first with theD1-like receptor antagonist SCH23390 for 15 min, followed byfenoldopam. SCH23390 abolished the stimulatory effect offenoldopam (21.2 � 5.5 pmol cAMP/mg protein). SCH23390did not have a significant effect when infused alone (19.5 � 1.9pmol cAMP/mg protein; Fig. 7).
Na�-K�-ATPase activity in response to D1-like receptorstimulation. Posterior gills of crabs infused with fenoldopamshowed a significant decrease in Na�-K�-ATPase activitycompared with vehicle infusion [fenoldopam � 13.3 � 0.9Pi·mg protein�1·h�1 vs. vehicle (control) � 24.6 � 1.9 �molPi·mg protein�1·h�1; Fig. 8]. To show that the decrease inactivity was due to D1-like dopamine receptors, the D1-likereceptor antagonist SCH23390 was infused alone or withfenoldopam. The infusion of SCH23390 alone did not affectthe activity of Na�-K�-ATPase (SCH23390 � 22.8 � 1.2�mol Pi·mg protein�1·h�1) but abolished the decrease in theactivity 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 ATPaseactivity among the groups, indicating that the activity ofATPases other than Na�-K�-ATPase was similar in all groups(data not shown). Fenoldopam infusion did not produce anychange in the expression of the Na�-K�-ATPase �-subunit(Fig. 9), indicating that the decrease in Na�-K�-ATPase ac-tivity was not related to the decreased expression of theprotein.
Fig. 5. Colocalization of Na�-K�-ATPase �-subunit and D1�R in posterior gills. Posterior gill sections of the blue crabs C. sapidus were immunostained forNa�-K�-ATPase �-subunit (Alexa 488, green) and D1�R (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 cellswithin the osmoregulatory patch and in cells near the tips of the lamellae. Scale bar � 50 um.
Fig. 6. Effect of fenoldopam on the expression of D1�R. Blue crabs(C. sapidus) in artificial seawater (10 ppt salinity) for 6 days were infused withvehicle plus fenoldopam or with vehicle alone (control). Posterior gills 6 and7 were excised, and whole gill tissue homogenate was prepared, resolved via10% SDS-PAGE, and probed with polyclonal D5R antibody and with goatanti-rabbit secondary antibody conjugated with horseradish peroxidase. Bandswere visualized using chemiluminescence. Actin was used as housekeepingprotein. Numerical data are expressed as means � SE. Fenoldopam-infusedcrabs showed a small, but significant, decrease in expression compared withcontrol (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 corre-sponding bar graphs.
R638 D1-LIKE DA RECEPTORS INHIBIT THE BLUE CRAB SODIUM PUMP
This study reports the presence of a D1-like dopaminereceptor, which is most likely the D1�R, in the posterior gillsof the Atlantic blue crab C. sapidus. When D1-like dopaminereceptors were stimulated with the D1-like receptor agonistfenoldopam in intact crabs, the posterior gills responded withan increase in cAMP production and a decrease in Na�-K�-ATPase activity without a change in expression of its �-sub-unit. Previous studies have indicated the presence of a dopa-mine receptor in crustaceans (14, 21, 23, 37, 46). However,among crustaceans, it is only in the spiny lobster P. interruptusthat two D1-like receptors, the D1�R and D1�R, have beendescribed so far (14). In this study, an 55-kDa band wasvisualized in the gills, which conceivably corresponds to thecrab D1�R. RT-PCR on cDNA prepared from the posteriorgills also indicated the presence of D1�R transcript.
Compared with previous studies, the present study employedtwo novel techniques to study the physiological response of the
dopamine receptor in the posterior gills of blue crabs. First, theexperiments in the current study were performed on livingcrabs and not on isolated gills. It is difficult to examine the invivo effects of perfused drugs in isolated gills of C. sapidussince properly securing a closed circuit system necessitatesclamping of the base of the gill where a large portion of the iontransporting cells are located (1, 32). Second this study used acontinuous drug infusion technique that allows for continuousbioavailability of the drug and a more stable pharmacokineticprofile (17, 49). In contrast, previous studies have employedthe technique of administering a single bolus of a pharmaco-logical agent into the crabs (37, 44, 45).
The dopamine receptor and the sodium pump colocalizedalong the basolateral membrane, especially in cells within theosmoregulatory patch. In addition to showing a potential forthe interaction between the sodium pump and the dopaminereceptor, these results suggest that the dopamine receptor mayalso play an important role in the regulation of sodium uptakein the posterior gills that may be associated with the osmoreg-ulatory response. Acute treatment with the D1-like receptoragonist fenoldopam resulted in a slight decrease in D1�Rexpression, which may be an acute compensatory mechanismto osmoregulate in lower salinities. In addition, the decrease inexpression may indicate receptor degradation, similar to whatoccurs 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 D1�R (D5R in mammals) is coupled to stimulatoryGs� and Gq�, both of which increase cAMP production (14).In contrast, the D2�PanR, a D2-like dopamine receptor, iscoupled to inhibitory G�i/�o, which decreases cAMP produc-tion (13, 14). Thus changes in the level of intracellular cAMPcan be used to monitor the physiological response of thedopamine receptors upon agonist activation. In the currentstudy, the stimulation of the D1-like receptors using fenoldo-pam led to an increase in cAMP production, indicating that theD1-like receptor was functional and that its signal transductionpathway was intact in the posterior gills.
Treatment with fenoldopam led to a decrease in Na�-K�-ATPase activity without a concomitant change in the expres-sion of the Na�-K�-ATPase-�-subunit expression. The Na�-K�-ATPase expression data suggest that the decrease in activ-ity was not due to the degradation of the transporter but ratherto an alteration of the intrinsic conformation of the sodiumpump, resulting in its internalization and inactivation. It isconceivable that the activation of Na�-K�-ATPase observed inprevious studies using high concentrations of dopamine,
Fig. 9. Effect of fenoldopam on the expression of Na�-K�-ATPase �-subunitin gills. Blue crabs (C. sapidus) transferred to artificial seawater (10 pptsalinity) for 6 days were infused with vehicle plus fenoldopam, or with vehiclealone (control). Posterior gills 6 and 7 were excised, and whole gill tissuehomogenates were prepared, resolved via 10% SDS-PAGE, and probed forNa�-K�-ATPase and actin (used as a housekeeping protein). Fenoldopam-infused crabs showed no change in Na�-K�-ATPase expression (n � 4)compared with control (n � 3).
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 wereinfused with vehicle plus the indicated drug or with vehicle alone (control).Posterior gills 6 and 7 were excised, and the whole gill homogenates wereassayed for cAMP (normalized for total protein concentration). Numerical dataare 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 thestimulatory 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.
Fig. 8. Na�-K�-ATPase activity in gills in response to drug infusion. Bluecrabs (C. sapidus) transferred to artificial seawater (10 ppt salinity) for 6 dayswere infused with vehicle plus indicated drug or with vehicle alone (control).Posterior gills 6 and 7 were excised and the whole gill homogenates wereassayed for Na�-K�-ATPase activity (normalized for the total protein con-centration). Numerical data are expressed as means � SE. Fenoldopam, aD1R/D5R agonist, significantly decreased Na�-K�-ATPase activity relative tocontrol. SCH23390, a D1R/D5R antagonist that by itself had no effect, blockedthe effect of fenoldopam on Na�-K�-ATPase activity. For abbreviations, seeFigure 7. *P 0.05 vs. other treatments, one-way ANOVA followed byHolm-Sidak posttest, n � 8–12/group.
R639D1-LIKE DA RECEPTORS INHIBIT THE BLUE CRAB SODIUM PUMP
fenoldopam, and SCH23390 is not due to the activation of theD1-like dopamine receptors alone but to the concomitant acti-vation of other receptors, e.g., serotonin receptors (vide infra).The role of D2-like receptors in the osmoregulation of euryha-line crabs should be also considered because it has been shownthat the perfusion of their posterior gills with spiperone anddomperidone, two potent D2-like dopamine receptor antago-nists, blocked the ability of dopamine to increase the transep-ithelial potential difference (21). Previous studies have mostlyused the natural ligand for the dopamine receptors, dopamine,as agonist; hence, the activation of the D2-like dopaminereceptors is likely to occur in this context since dopamine isable to activate G�s, G�q, and G�i in crustaceans (13, 14).Under certain circumstances, the mammalian D3R, a D2-likedopamine receptor, can be linked to G�s (40). Thus it ispossible that an increase or a decrease in cAMP can ensue afterdopamine activation, depending on which cascade dominatesthe response. However, what regulates the eventual responsemust be determined.
Previous studies have shown that an increase in cAMPproduction in the osmoregulatory gills of euryhaline crusta-ceans is associated with an increase in Na�-K�-ATPase activ-ity and a transient activation followed by a subsequent inhibi-tion of transepithelial potential difference (21, 23, 44). Theseauthors suggested that the transient increase in cAMP contentwas responsible for Na�-K�-ATPase stimulation. However,direct proof of this hypothesis has not been forthcoming. Itwould be interesting to determine whether D1- and D2-likereceptors interact in the regulation of Na�-K�-ATPase incrustacean gills. This occurs in mammalian renal tubule cellsunder conditions of moderate extracellular fluid expansion,where the D2-like receptors act synergistically with the D1-likereceptors to increase sodium excretion by inhibiting Na�-K�-ATPase activity (3, 16, 29).
In mammals, higher concentrations of dopamine can activatenondopamine receptors, e.g., serotonin and adrenergic recep-tors. There are studies showing that D1-like receptor drugs,such as fenoldopam and SCH23390, have high affinities forserotonin receptors (12, 35), a possibility with the high con-centrations (10 �M) used in previous reports. Several crusta-cean serotonin receptors, which also increase cAMP produc-tion upon stimulation, have been reported in the somatogastricganglion but not in the gills; these crustacean serotonin recep-tors are not pharmacologically similar to vertebrate serotoninreceptors (14, 46, 47). Octopamine (OA), a biogenic mono-amine structurally related to norepinephrine, acts as a neuro-hormone, neuromodulator, and neurotransmitter in inverte-brates, including the crustaceans. Activation of the OA recep-tors, which are similar to the mammalian adrenergic receptors,also invariably results in increased cAMP levels (42). Thesereceptors, if proven to be present in the posterior gills, couldconceivably account for the previous observations subsequentto the treatment with dopamine.
Perspectives and Significance
In summary, we have provided evidence for the presence ofdopamine receptors, presumably the D1�R, which colocalizewith the Na�-K�-ATPase at the basolateral membrane of thecells that populate the osmoregulatory patch of the posteriorgills of the blue crab. While we have shown that agonist
stimulation of these receptors resulted in diminished receptorabundance, increased cAMP production, and decreased sodiumtransport via the inhibition of Na�-K�-ATPase activity, thedetails of the molecular mechanisms involved in this processhave yet to be established. The inhibition of Na�-K�-ATPaseactivity by the D1-like dopamine receptors appears to havebeen conserved in phylogenetically distinct organisms. Inmammalian renal tubule cells under conditions of moderatesodium excess, the D1-like dopamine receptors are responsiblefor decreasing renal sodium transport by at least 50% (10, 18,24, 50, 51). Mammalian renal tubule cells can adapt in anenvironment of low (50 mosmol/kgH2O) to high osmolality(1,200 mosmol/kgH2O) (30), similar to conditions to whicheuryhaline crustaceans are exposed during their migration fromlow- to high-salinity water. TonEBP (30), which plays animportant 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 inspecialized tissues is shared by various organisms belonging tovarious phyla; however, unique variations in terms of novelinteracting proteins or novel functions for the same proteinexist to allow the organisms to thrive in their own biologicalniche. It would be interesting to determine if the D1- andD2-like receptors interact in the regulation of Na�-K�-ATPasein crustacean gills and establish the cross talk between dopa-mine and other hormones in osmoregulation. Understandingthe underlying mechanisms may shed light on the evolution ofthe pathways involved in ionic and osmotic regulation.
ACKNOWLEDGMENTS
We thank Dr. Deborah J. Baro, Georgia State University, for the rabbitanti-lobster dopamine receptor antibodies and immunizing peptides that wereused to verify the crab D1�R.
GRANTS
This work was supported in part by National Institutes of Health GrantsR01-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. concep-tion 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 experi-ments; 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 ofmanuscript; I.A. drafted manuscript.
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