isoform-like proteins in marine euryhaline milkfish (Chanos

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RELATIVE CHANGES IN THE ABUNDANCE OF BRANCHIAL 523produce diluted urine via the kidneys to balancethe passive water gain, and actively absorb saltsthrough the gills from the environment (Marshalland Grosell, 2006). Effective ionoregulatory mechanismsthus enable teleosts to retain an osmoticand ionic constancy in their internal milieus andsurvive in hypertonic or hypotonic environments.Na 1 /K 1 -ATPase (NKA) is a membrane-spanningprotein that couples the exchange of twoextracellular K 1 ions for three intracellular Na 1ions to the hydrolysis of one molecule of ATP (Postand Jolly, ’57). It is crucial not only for sustainingintracellular homeostasis, but also for providing adriving force for many transporting systemsincluding fish gills (Hwang and Lee, 2007). Thea-subunit of NKA containing the binding sites forATP, Na 1 , and K 1 is considered to be the catalyticsubunit. The b-subunit is a type II glycosylatedpolypeptide that is thought to assist in the foldingand placement of the a-subunit into the cellmembrane (Blanco and Mercer, ’98). A third,nonessential g-subunit has been identified inmammals (Reeves et al., ’80) and is thought toplay roles in modulating Na 1 , K 1 , and ATPbinding affinities to the NKA ab complex (Therienet al., ’99). Immunocytochemical studies demonstratedthat NKA was localized mainly in epithelialmitochondrion-rich cells of teleostean gills.The current model depicted that mitochondrionrichcells in gill epithelia of teleosts used NKA asthe primary driving force for operation of thedifferent ion transporters depending on theenvironmental salinity (Hwang and Lee, 2007;Evans, 2008).NKA exhibits four a (a1-4) and three b (b1-3)isoforms. Sequence variations in the amino terminusand 11 amino acids in the large intracellularloop between transmembrane domains 4 and 5distinguished four isoforms of the NKA a-subunit(Takeyasu et al., ’90; Pressley, ’92). Thesea-subunit isoforms, which showed tissue-specificand developmentally dependent patterns of expressionin birds and mammals, have beensuggested to be presented in all vertebrate classes,including teleosts (Takeyasu et al., ’90; Pressley,’92). Comparing the sequences of one orthologousisoform from many different species yields anamino acid identity of 90% or greater (Blanco andMercer, ’98). In teleosts, full length sequence ofNKA a-isoforms have been cloned in the whitesucker (a1; Catostomus commersoni; Schönrocket al., ’91), the European eel (a; Anguilla anguilla;Cutler et al., ’95), tilapia (a1 and a3; Oreochromismossambicus; Feng et al., 2002), killifish (a1 anda2; Fundulus heteroclitus; Semple et al., 2002),and rainbow trout (a1a, a1b, a1c, a2, and a3;Oncorhynchus mykiss; Richards et al., 2003). Inaddition, nine distinct NKA a-subunit genes havebeen identified in zebrafish (Danio rerio) genomicdatabases. Among them, four (a1a.1, a1a.2, a1a.4,and a1a.5) of the seven a1 genes were found to beexpressed in the mucous cells of skin (Blasioleet al., 2002; Canfield et al., 2002). In addition,partial sequences of a-isoforms were reported inthe spiny dogfish (a3; Squalus acanthias; Hansen,’99) and the Antarctic nototheniid (a1, a2, and a3;Trematomus bernachii; Guynn et al., 2002).Milkfish (Chanos chanos) is a marine teleostwidely distributed throughout the tropical andsubtropical Indo-Pacific. The euryhalinity of milkfishwas demonstrated throughout its life history,although it did not appear to require the freshwater (FW) for any part of its life cycle (Bagrinao,’94). Milkfish are found naturally or culturedcommercially in FW, SW, and hypersaline water(HSW) (Crear, ’80). These characteristics makemilkfish a good experimental species for studies onosmoregulation. Previous studies revealed thatmilkfish, like other euryhaline teleosts, exhibitedadaptive changes in branchial NKA responses (i.e.,mRNA, protein, activity, immunoreactive cellnumber) upon salinity challenge (Lin et al., 2003,2006). Moreover, Pagliarani et al. (’91) demonstratedthat NKA proteins isolated from FW- andSW-acclimated fish gills had different biochemicalproperties and suggested that different NKAisoforms might correlate with ion regulation indifferent environmental salinities. Since a-subunitis the catalytic subunit of NKA, alteration of NKAa-isoforms protein expression was supposed to be acrucial mechanism for euryhaline teleosts to adaptto environments of various salinities. Feng et al.(2002) provided evidence of enhanced transcriptionof branchial NKA a1 and a3 genes inMozambique tilapia following transfer from FWto SW. Recent studies focused on mRNA expressionpatterns of two NKA a-isoforms (a1a and a1b)revealed their different responses to salinitychanges in salmonid fishes (Richards et al., 2003;Shrimpton et al., 2005; Bystriansky et al., 2006,2007a,b; Nilsen et al., 2007). However, Morrisonet al. (2006) reported that changes in the protein,but not mRNA levels of NKA a-subunit isoforms(i.e., a1, a2, and a3), occurred in the gills of warmacclimated Antarctic teleost (Trematomus bernacchii).This motivates this study to investigate theprotein expression patterns of gill NKA a-subunitisoforms in euryhaline milkfish following salinity-J. Exp. Zool.
524C.-H. TANG ET AL.transfer, to illustrate the potential role of NKAa-isoforms in this marine euryhaline teleost uponsalinity challenge.MATERIALS AND METHODSExperimental animalsJuvenile milkfish (C. chanos) with 13.670.2 cmtotal length and 22.175.9 g body weight wereobtained from a local fish farm. Values of total lengthand body weight were expressed as the mean7SEM.SW (35%) orHSW(60%) were prepared from localtap water (i.e., FW) added with proper amounts ofsynthetic sea salt (Instant Ocean, Aquarium Systems,Mentor, OH). The water was continuously circulatedthrough fabric-floss filters. Milkfish were kept in SWat 28711C with a daily 12 hr photoperiod for at leasttwo weeks before experiments. Fish were fed a dailydiet of commercial fodder.Acclimation experimentsMilkfish kept in the laboratory for at least twoweeks were transferred into experimental tankswith HSW, SW, and FW, respectively, and theculture conditions were identical to thosedescribed above. Milkfish were held in experimentaltanks for two weeks prior to sampling.Time-course environmentsFor time-course experiments, milkfish of theexperimental group were transferred directly fromstock SW to FW or HSW, while fish in the controlgroup were transferred to SW simultaneously.Because fish were transferred by nets, the controlgroup was used to monitor the effect of handlingfish alone. After transfer, the fish were caught bynetting at 12, 24, 48, 96, and 168 hr and sampledfor immunoblotting.Preparation of gill homogenatesGills were excised and blotted dry. After removingthe gill arches, gills were immediately cut intosmall pieces. All subsequent procedures wereperformed on ice. Gill scrapings were suspendedin the mixture of homogenization solution (100 mMimidazole-HCl buffer, pH 7.6; 5 mM Na 2 EDTA;200 mM sucrose; and 0.1% sodium deoxycholate)and proteinase inhibitors (10 mg antipain, 5 mgleupeptin, and 50 mg benzamidine dissolved in5 mL aprotinin) (v/v: 100/1). Homogenization wasperformed with a homogenizer (Polytron PT1200E KINEMATICA, Lucerne, Switzerland) at amaximum speed for 25 strokes. The homogenateswere then centrifuged at 13,000g for 20 min at 41C.The supernatants were used to determine proteinconcentrations and for immunoblotting. Proteinconcentrations were identified by BCA proteinassay reagents (PIERCE, Rockford, IL) usingbovine serum albumin (Sigma, St. Louis, MO) asa standard. All gill lysates were stored at 801Cbefore immunoblotting.AntibodiesNKA a1-isoform antibody, mouse monoclonalantibody (a6F) raised against the a1-isoform of theavian NKA was purchased from the DevelopmentalStudies Hybridoma Bank (DSHB; Iowa City,IA). The dilution rate is 1:5,000 for immunoblotting.NKA a2-isoform antibody was the commercialgoat polyclonal antibody (sc-16049), raisedagainst a peptide mapping within an internalregion of the NKA a2-subunit of human origin(Santa Cruz Biotechnology, Santa Cruz, CA). Thedilution rate is 1:500 for immunoblotting. NKAa3-isoform antibody was the commercial goatpolyclonal antibody (sc-16052), raised against apeptide mapping within an internal region of NKAa3 of human origin (Santa Cruz Biotechnology).The dilution rate is 1:100 for immunoblotting.Alkaline phosphatase-conjugated goat anti-mouseor rabbit anti-goat antibodies (Jackson ImmunoResearch, West Grove, PA) were used as secondaryantibodies for immunoblotting.Immunoblotting analysisImmunoblotting procedures were performed accordingto Tang et al. (2008) with little modification.Seventy-five microgram of protein from gillhomogenates was heated with sample buffer at371C for 30 min and fractionated by electrophoresison sodium dodecyl sulfate-containing 7.5% polyacrylamidegels. The prestained protein molecularweight marker was purchased form Fermentas(SM0671; Hanover, MD). Separated proteins werethen electro-ransferred to PVDF membranes(Millipore, Bedford, MA). Blots were preincubatedin PBST (phosphate buffer saline with Tween 20)buffer (137 mmol L 1 NaCl; 3 mmol L 1 KCl;10 mmol L 1 Na 2 HPO 4 ;2mmolL 1 KH 2 PO 4 ;0.2%Tween 20; pH 7.4) containing 5% w/v nonfat driedmilk to minimize nonspecific binding, then incubatedat 41C overnight, with the primary antibodiesdiluted in PBST containing 1% BSA and 0.05%sodium azide. The blots were washed in PBST andincubated at room temperature for 1 hr withJ. Exp. Zool.
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isoform-like proteins in marine euryhaline milkfish (Chanos

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