Alex M. Zimmer C. Michele Nawata Chris M. Wood. than Na? influx itself, is critical to the facilitation of ammonia excretion.

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1 DOI /s ORIGINAL PAPER Physiologicl nd moleculr nlysis of the interctive effects of feeding nd high environmentl mmoni on rnchil mmoni excretion nd N + uptke in freshwter rinow trout Alex M. Zimmer C. Michele Nwt Chris M. Wood Received: 15 Ferury 2010 / Revised: 11 My 2010 / Accepted: 4 June 2010 Ó Springer-Verlg 2010 Astrct Recently, N? /NH? 4 exchnge complex model hs een proposed for mmoni excretion in freshwter fish. The model suggests tht mmoni trnsport occurs vi Rhesus (Rh) glycoproteins nd is fcilitted y gill oundry lyer cidifiction ttriutle to the hydrtion of CO 2 nd H? efflux y N? /H? exchnger (NHE-2) nd H? -ATPse. The ltter two mechnisms of oundry lyer cidifiction would occur in conjunction with N? influx (through N? chnnel energized y H? -ATPse nd directly vi NHE-2). Here, we show tht nturl mmoni loding vi feeding increses rnchil mrna expression of Rh genes, NHE-2, nd H? -ATPse, s well s H? -ATPse ctivity in juvenile trout, similr to previous findings with mmonium slt infusions nd high environmentl mmoni (HEA) exposure. The ssocited increse in mmoni excretion occurs in conjunction with fourfold increse in N? influx fter mel. When exposed to HEA (1.5 mmol/l NH 4 HCO 3 t ph 8.0), oth unfed nd fed trout showed differentil increses in mrna expression of Rhcg2, NHE-2, nd H? -ATPse, ut H? -ATPse ctivity remined t control levels. Unfed fish exposed to HEA displyed chrcteristic reversl of mmoni excretion, initilly uptking mmoni, wheres fed fish (4 h fter the mel) did not show this reversl, eing le to immeditely excrete mmoni ginst the grdient imposed y HEA. Exposure to HEA lso led to depression of N? influx, demonstrting tht mmoni excretion cn e uncoupled from N? influx. We suggest tht the efflux of H?, rther Communicted y I. D. Hume. A. M. Zimmer (&) C. M. Nwt C. M. Wood Deprtment of Biology, McMster University, Hmilton, ON L8S 4K1, Cnd e-mil: zimme@mcmster.c thn N? influx itself, is criticl to the fcilittion of mmoni excretion. Keywords Rhesus glycoproteins Ammoni excretion High environmentl mmoni (HEA) Rinow trout Feeding Arevitions HEA High environmentl mmoni J mm Ammoni flux N J in Sodium influx N J out Sodium efflux N J net Sodium net flux mrna Messenger RNA NHE-2 N? /H? exchnger-2 P NH3 Prtil pressure of NH 3 Rh Rhesus glycoprotein Totl mmoni concentrtion T mm Introduction In rinow trout (Oncorhynchus mykiss), s well s in mjority of other freshwter teleost fish, lrge proportion of nitrogenous wste is excreted s mmoni vi the gills with reltively smll mount eing excreted renlly (Smith 1929; Fromm 1963; Wood 1993). Ammoni excretion increses fter mmoni loding vi direct infusion (Metz 1972; Wilson et l. 1994; Slm et l. 1999; Nwt nd Wood 2009) nd is inhiited y exposure to high environmentl mmoni (HEA), ut with lter recovery (Fromm nd Gillette 1968; Pyn 1978; Wilson et l. 1994; Nwt et l. 2007). These tretments re

2 ssocited with increses in N? influx from the wter during mmoni infusion (Wilson et l. 1994; Slm et l. 1999) nd decreses in N? influx nd lter recovery during HEA (Twitchen nd Eddy 1994; Wilson et l. 1994). A reltively new ide is the potentil involvement of Rh proteins in these processes (Weihruch et l. 2009; Wright nd Wood 2009). Rhesus (Rh) proteins re thought to e implicted in mmoni trnsport due to their similrity to the methylmmonium/mmonium permese (MEP)/mmoni trnsporter (Amt) protein fmilies (Mrini et l. 1997). It remins controversil whether they trnsport NH 3 or NH? 4 (Khdemi et l. 2004; Bkouhetl. 2004; Lietl. 2007). Recently, severl Rh cdna sequences were identified in the gills of rinow trout (Nwt et l. 2007), mngrove killifish (Hung et l. 2007), pufferfish (Nkd et l. 2007), nd zerfish (Nkd et l. 2007). When expressed in Xenopus oocytes, trout Rh proteins significntly incresed the flux of mmoni, with experimentl results fvoring the trnsport of NH 3 rther thn NH? 4 (Nwt et l. 2010). Furthermore, fish exposed to HEA or infused with mmoni displyed increses in rnchil mrna levels of Rhcg2 s well s N? /H? exchnger (NHE-2) nd/or V- type H? -ATPse (Hung et l. 2007; Nwt nd Wood 2009; Nwt et l. 2007). Tsui et l. (2009) demonstrted tht, in vitro, gill cells of rinow trout pre-exposed to HEA nd cortisol show increses in Rhcg2, H? -ATPse, nd NHE-2 gene expression. The prtil pressure grdient for NH 3 (P NH3 ), which is thought to drive mmoni excretion, ppers to e mintined y the cidifiction of the gill oundry lyer (Avell nd Bornncin 1989; Rndll nd Wright 1987; Wilson et l. 1994; Slm et l. 1999). This cuses protontion of NH 3 fter it diffuses cross the gills, trnsforming it to NH? 4 nd thus mintining positive P NH3 grdient from lood to wter under norml conditions (see Wood 1993). Acidifiction of the gill oundry lyer is thought to e due in prt to n piclly locted V- type H? -ATPse (Lin et l. 1994; Sullivn et l. 1995), n picl H? /N? exchnger (NHE; Wilson et l. 2000; Ivnis et l. 2008), nd lso the hydrtion of CO 2 (see Wilkie 2002). In freshwter fish, ctive pumping of H? out of the gills y the picl H? - ATPse is elieved to crete n electrochemicl grdient, which drives N? into the gills (Avell nd Bornncin 1989; Lin nd Rndll 1991, 1993; Wilson et l. 2000). Apicl NHEs my lso function in N? uptke in conjunction with solterlly locted N? /K? -ATPse nd n electrogenic N? HCO - 3 cotrnsporter (NBC-1) (Wilson et l. 2000; Perry et l. 2003; Scott et l. 2005; Prks et l. 2007). Thus, the gill oundry lyer cidifiction tht fcilittes the trnsport of mmoni would e linked to N? influx in this scheme. Recently, Wright nd Wood (2009) proposed n extension of this model for mmoni excretion in freshwter fish, N? /NH? 4 exchnge complex, which ws sed minly on dt of Tsui et l. (2009) otined from cultured gill epitheli in vitro. The model suggests tht mmoni moves pssively through Rhcg (presumly Rhcg2 in rinow trout) s NH 3 nd is coupled to the efflux of H? through H? -ATPse nd NHE-2. These mechnisms my provide the driving force necessry for N? uptke (through NHE-2 or through N? chnnel energized y H? -ATPse), ut my not necessrily do so in 1:1 rtio. Very recently, prtil support for this model ws provided y Wu et l. (2010) from in situ studies of mitochondrilrich cells (MRCs) in the lrve of Jpnese medk. Here mmoni excretion through Rhcg (Rhcg1) ws dependent on H? excretion y NHE-3 nd ppered to e tightly linked to N? uptke. However, in this cse, H? flux ws shown to e insensitive to filomycin A1, demonstrting tht H? -ATPse does not necessrily ply role (Wu et l. 2010). This work contrsts with the findings of Nkd et l. (2007) tht Rhcg1 co-loclizes with V H? -ATPse nd Rhcg1 in MRCs in the skin of lrvl zerfish. These studies demonstrte the complexity nd vriility of the reltionship mong different trnsport proteins nd different species. However, to dte, wht hs een missing is ny exmintion in intct nimls of the potentil linkges of mmoni flux vi Rh proteins to N? influx vi NHE or H? -ATPse. Also overlooked to dte is the potentil role of feeding, s mechnistic in vivo studies re trditionlly done on fsted fish to stndrdize N sttus. Fromm (1963) showed tht during unfed periods, totl nitrogenous wste excretion of trout flls sustntilly to seline level. It is now well estlished tht plsm totl mmoni concentrtion (T mm ) nd mmoni excretion rte increse mrkedly fter feeding in fish (Kushik nd Teles 1985; Wicks nd Rndll 2002, ; Wood 1993; Gelineu et l. 1998; Bucking nd Wood 2008). Bsed on findings with HEA nd mmoni infusion discussed ove, we hypothesized tht this more nturl pthwy of internl mmoni loding in trout would cuse upregultion of Rhcg2, NHE-2, nd H? -ATPse mrna in the gills nd incresed N? influx fter feeding. This study ims to determine the effects of feeding, HEA exposure, nd comintion of oth feeding nd HEA on mmoni excretion, plsm T mm,n? uptke, nd gill mrna levels of Rhcg2, NHE-2, nd H? ATPse in juvenile rinow trout, s well s rnchil H? ATPse ctivity. Specificlly, we hypothesized tht fed fish under norml conditions (no HEA) would demonstrte increses in mmoni excretion, plsm T mm, N? uptke, nd mrna expression of ssocited gill trnsport proteins. Secondly, we hypothesized tht this prior upregultion would llow fed fish to excrete mmoni ginst HEA etter thn unfed fish. Thirdly, we predicted tht HEA would inhiit N? influx in oth fed nd unfed fish, ut

3 recovery of oth N? influx nd mmoni excretion would e more rpid in fed fish since they would lredy hve incresed levels of Rhcg2, NHE2, nd H? ATPse Methods nd mterils Animls J mm (μmol/g/h) Juvenile rinow trout (Oncorhynchus mykiss) weighing 3 9 g were otined from Humer Springs Trout Htchery, Ontrio, Cnd. The fish were kept in dechlorinted Hmilton tp wter (modertely hrd: [N? ] = 0.6 mequiv/l, [Cl - ] = 0.8 mequiv/l, [C 2? ] = 1.8 mequiv/l, [Mg 2? ] = 0.3 mequiv/l, [K? ] = 0.05 mequiv/l; titrtion lklinity 2.1 mequiv/l; ph 8.0; hrdness 140 mg/l s CCO 3 equivlents; temperture C), nd fed 1% of their weight per dy with commercil trout pellets. The mesured N? nd Cl - contents of the pellets were pproximtely 200 mequiv/kg ech. Fish were fsted for 48 h prior to the strt of experiments. Experimentl series The study consisted of vriety of experiments, ll performed t ph of 8.0, with ech fish plced in n individul 80-ml chmer contining erted wter t pproximtely 15 C nd limited light exposure to reduce stress. The tretments included exposure to high environmentl mmoni (HEA; 1.5 mm NH 4 HCO 3 t ph 8.0), feeding to stition, feeding to stition followed y HEA exposure, nd no feeding nd no HEA exposure. The ph ws monitored throughout using Rdiometer-Copenhgen comintion glss electrode (GK24O1C) nd mintined y the ddition of 0.1 M KOH or 0.1 M HCl to the chmers s necessry. Ammoni excretion experiments A vriety of experiments designed to evlute mmoni excretion were performed. Exposures hd durtions of 6, 12, or 24 h. Those lsting longer thn 6 h were flushed with new wter every 6 h to limit mmoni uildup in the chmers. For mesurements of mmoni excretion rtes, wter smples (2 ml) were tken t 2-h intervls (in one experiment t 3-h intervls). For fed tretments, trout were fed to stition in their communl holding tnks (they would not feed in the individul chmers) nd then trnsferred immeditely to the chmers. For n initil 12-h post-prndil evlution of mmoni excretion, flux mesurements were strted immeditely fter trnsfer. Bsed on this initil test (see Fig. 1), s well s the study of Gelineu et l. (1998), in susequent experiments, fed fish egn the HEA or corresponding Control unfed Fig. 1 Effects of feeding on mmoni excretion rtes (J mm ) over 12 h including rtes from 6-h control using n unfed group of fish. Feeding occurred t 0 h. Mens shring the sme letter re not significntly different from one nother. Comprisons were mde using one-wy ANOVA sttisticl nlysis followed y Fisher LSD post hoc multiple comprison test (P \ 0.05, N = 8) control exposures t 4 h fter feeding (i.e., 3? h fter trnsfer) s this seemed to e the time t which elevted mmoni excretion nd, potentilly, trnsporter upregultion, would e well estlished in trout. Therefore, for these HEA exposures nd corresponding control exposures (no HEA), oth fed nd unfed fish were cclimted to the chmers for t lest 3 h prior to the strt of the exposures. Plsm mmoni nd tissue collection experiments Fish were exposed to the sme conditions s mentioned ove. Fed nd unfed fish were euthnized t times corresponding to 2, 4, 6, 8, nd 10 h post-feeding. Trout were killed using n MS-222 solution (0.1 g/l), which hd een set to the sme ph nd mmoni concentrtion s the experimentl conditions. Blood smples were collected into sodium-heprinized plstic microhemtocrit cpillry tues from the cudl vessels fter severing the til, nd plsm ws seprted from red cells y centrifugtion (10,000g for 2 min). Though this method of collection hd the possiility of rising plsm T mm (see Discussion ), the effect would likely e uniform cross tretments. Plsm smples were susequently flsh-frozen in liquid nitrogen nd stored t -70 C. Gill (entire gill sket) smples were dissected from the fish following lood smpling nd were immeditely flsh-frozen in liquid nitrogen nd stored t -70 C until nlysis. Sodium flux experiments Experiments to determine sodium flux were performed seprtely from ll other experiments. Fish were plced c c

4 into identicl chmers (t pproprite times post-feeding) s descried ove nd 1 lci/l of 22 N ws dded to ech chmer. When chmers were flushed t 6-h time periods, rdioisotope ws re-dministered to ech chmer. All other procedures were performed identiclly to the mmoni excretion experiments descried ove. However, terminl plsm collection ws done using mmonium-heprinized glss microhemtocrit cpillry tues. These smples were required for mesurement of finl plsm 22 N nd totl N? levels, for ckflux correction (see elow). Determintion of mrna expression Totl RNA ws extrcted from gill smples using TRIzol (Invitrogen, Burlington, ON), quntified vi spectrophotometry (Nnodrop ND-1000, Nnodrop Technologies, Wilmington, DE, USA) nd electrophoresed on 1% grose gels stined with ethidium romide to verify integrity. cdna ws synthesized from 1 lg totl RNA (DNseItreted, Invitrogen) with n oligo(dt 17 ) primer nd Superscript II reverse trnscriptse (Invitrogen). mrna expression levels of Rhcg2, H? -ATPse (v-type, B-suunit), nd NHE2 in the gill were determined y quntittive rel-time PCR (qpcr) using the ove synthesized cdna nd trnsporter-specific primers pulished previously (Nwt et l. 2007). Anlyses were performed on n Mx3000P QPCR System (Strtgene, Cedr Creek, TX). Rections (20 ll) contining 4 ll of 59 diluted cdna descried ove, 4 pmol ech of forwrd nd reverse primers, 0.8 ll ROX dye (1:10 dilution), nd 10 ll Pltinum SYBR Green qpcr SuperMix-UDG (Invitrogen), were performed t 50 C (2 min) nd 95 C (2 min), followed y 40 cycles of 95 C (15 s) nd 60 C (30 s). No-templte controls nd nonreversed-trnscried controls were run in prllel while melt-curve nlysis verified the presence of single product. Vlues were extrpolted from stndrd curves generted y the seril dilution of one rndomly selected control smple. Becuse the mrna expression levels for three housekeeping genes, et-ctin, elongtion fctor-1, nd 18S, were unstle cross tretments, dt were normlized to ng totl RNA, n estlished method of normliztion (Bustin 2000, 2002), which we hve used previously for these genes in trout gills (Nwt nd Wood 2008, 2009). Determintion of rnchil H? - ATPse ctivity Gill H? -ATPse ctivity mesurements were otined from the sme unperfused tissue smples (control, fed no HEA, fed nd unfed HEA) used for mrna expression mesurements. A protocol for mesuring H? -ATPse ctivity ws modified from Lin nd Rndll (1993) nd dpted to N? /K? -ATPse ctivity ssy (McCormick 1993) using sodium zide nd NEM (N-ethylmleimide) nd ouin s inhiitors to mesure H? -ATPse ctivity levels in control nd experimentl fish gills. This ws the sme protocol used y Nwt et l. (2007). Activities re reported s ctivity (no inhiitor) minus inhiitor-treted ctivity (sodium zide with N-ethylmleimide (NEM) for V-type H? -ATPse), mesured t 340 nm nd t room temperture in kinetic microplte reder (SpectrMAX Plus; Moleculr Devices, Menlo Prk, CA) t 15-s intervls for 30 min. Protein concentrtions were mesured using Brdford Regent (Sigm) nd BSA stndrds. Anlyticl techniques For plsm totl mmoni (T mm ) nlyses, freshly thwed plsm smples were ssyed enzymticlly using Richem kit (Cliniq Corportion, Sn Diego, CA). For wter totl mmoni nlyses, the collected wter smples were refrigerted t -4 C for no more thn 24 h or frozen (-20 C) until ssy. Wter mmoni concentrtion ws determined (in triplicte) vi spectrophotometry using modified protocol sed on indophenol formtion with sodium slicylte (Verdouw et l. 1978). The two different mmoni ssys were cross-vlidted. Gmm-rdioctivity of 22 N (in counts per minute, cpm) ws mesured y gmm counting (Perkin Elmer Wizrd Auto Gmm Counter) nd sodium concentrtion ws mesured y tomic sorption spectroscopy (SpectrAA 220FS Atomic Asorption Spectrometer). Clcultions Ammoni flux rtes (lmol/g/h) were clculted s: J Amm ¼ ½AmmŠ i ½Amm V= ðt MÞ; ð1þ Š f where [Amm] i nd [Amm] f re the initil nd finl concentrtions of mmoni in the wter (in lmoles/l) otined from comprison to stndrd curve. V indictes volume (l), t time (h), nd M mss (g). Sodium influx (lmol/g/h) ws mesured using vlues otined from gmm counting (cpm/ml), nd wter nd plsm concentrtions (lmol/ml) otined from tomic sorption spectroscopy. The following eqution descried y Metz (1956), which incorportes ckflux corrections, ws used: ¼ ð Ri Rf ÞV ½ ext SA int ð½nš i ½N f Þ = J N in ½ðSA ext SA int ÞM tš; ð2þ where Ri nd Rf re the initil nd finl rdioctivity levels (cpm/ml) in the wter for ech flux period, V ext indictes finl volume (ml) of wter, SA int nd SA ext re the men internl nd externl specific ctivities over the flux period, nd [N] i nd [N] f re the initil nd finl concentrtions

5 of sodium (in lmol/ml) mesured y tomic sorption. Net sodium flux (lmol/g/h) ws otined using the following eqution: J N net ¼ ½N Š i ½ N Š f V= ðm tþ: ð3þ Finlly, sodium efflux (lmol/g/h) ws otined using the following eqution: Jout N ¼ JN net JN in : ð4þ To clculte vlues for wter nd plsm mmoni chemistry ([NH? 4 ], [NH 3 ], nd P NH3 ), pk nd NH 3 constnts for the pproprite experimentl ph nd temperture were otined from Cmeron nd Heisler (1983). Using pk nd the experimentl T mm vlues, [NH? 4 ] ws derived from modified version of the Henderson Hssellch eqution: ¼ Tmm = ½1 þ ntilogðph pkþš ð5þ NH þ 4 From this, [NH 3 ] ws clculted from the following eqution: ½NH 3 Š ¼ T Amm NH þ 4 ð6þ Then P NH3 (in ltorr) ws clculted using the pproprite NH 3 vlues from Cmeron nd Heisler (1983) using the following eqution: P NH3 ¼ NH 3 =NH 3 ð7þ D P NH 3 vlues were clculted y sutrcting the corresponding P NH3 vlue clculted for wter from tht clculted for lood plsm t the sme time for ech individul fish. For these clcultions, lood plsm ph of 7.9 ws ssumed (see Discussion ). Sttisticl nlyses Dt hve een expressed s mens ± 1 SEM (N = numer of fish). In the cse of the 6-h mmoni flux (J mm ) experiments, controls re representtive of n verge of 6-h flux mde over the corresponding time period for trout sujected to no HEA, no feeding, or feeding only, s pproprite (see Results ). Comprisons etween these controls (significnt differences represented y sterisks) were mde using Mnn Whitney rnk sum test. Comprisons mde etween experimentl dt points nd given control were nlyzed using one-wy ANOVA followed y Dunnett s method post hoc test. In the cse of filed normlity test, n ANOVA on rnks nlysis ws performed using Dunn s post hoc test with multiple comprisons versus control. All comprisons mde mong multiple tretments within time points (significnce represented using the lettering system, wherey mens shring the sme letter re not significntly different) were nlyzed using one-wy ANOVA nlysis with Fisher LSD post hoc test. In the cse of filed normlity test, n ANOVA on rnks nlysis ws performed using Dunn s post hoc test with multiple pir-wise comprisons. In ll cses, sttisticl significnce ws ccepted t the P = 0.05 level. Figure legends specify the sttisticl test employed to determine significnce within the given dt. Results Control experiments (6 h) with unfed fish were performed in ll experimentl series s check on hndling disturnce. As mmoni excretion rtes nd N? flux rtes do not vry significntly over time, verge vlues re shown s men control vlues in figures. Ammoni excretion Prior to feeding, mmoni excretion (J mm ) ws typiclly round -0.4 lmol/g/h in juvenile trout, which hd een fsted for 48 h. Following feeding, J mm incresed 2.4-fold y 2 4 h, nd 3.6-fold y 8 10 h (Fig. 1). When exposed to HEA, unfed fish displyed reversl of mmoni flux from control (no HEA) vlue of lmol/g/h to?0.62 lmol/g/h (i.e., net mmoni uptke) over the first 3 h of exposure (Fig. 2). However, fter 3 h of HEA, these fish were le to re-estlish mmoni excretion to vlue not significntly different from the control, sitution which persisted through h despite the continued presence of HEA (Fig. 2). Therefore, in ll susequent experiments, fed fish were J mm (μmol/g/h) high externl mmoni Control unfed Fig. 2 Ammoni excretion rtes (J mm ) in unfed fish during HEA exposure including rtes from 6-h control using n unfed group of fish. HEA exposure egn t 0 h. Asterisks denote vlues tht re significntly different from the control vlue sed on one-wy ANOVA sttisticl nlysis followed y Dunnett s post hoc test ginst the control vlue (P \ 0.05, N = 6 8)

6 exposed to HEA t 4 10 h post-feeding, time y which the elevted J mm response ws well estlished, nd mesurements were tken of J mm t 2-h intervls commencing with the strt of HEA exposure, to cpture the time period over which J mm would e reversed nd mmoni loding would occur in unfed fish. A susequent experiment compred the J mm responses of unfed nd fed trout to HEA exposure t time corresponding to 4 10 h post-feeding (Fig. 3). The control vlues in Fig. 3 represent verges of 6-h control fluxes (no HEA) over the pproprite period for fed nd unfed fish, respectively. Note tht the fed control vlue (-0.88 lmol/ g/h) ws significntly greter thn the unfed control vlue (-0.40 lmol/g/h), reinforcing the oservtion tht feeding elevtes J mm (Fig. 3). As in the erlier series (Fig. 2), unfed fish gin experienced reversl of J mm over the first 2 h of HEA, with susequent return to control rtes therefter. In contrst, fed fish exposed to HEA (4 h postfeeding) displyed no significnt chnges from the control over 6 h of exposure (Fig. 3), therey mintining J mm in the fce of HEA. Plsm totl mmoni levels Feeding lone with no HEA induced mrked, ut delyed increse of plsm totl mmoni (T mm ) from 555 lmol/l in the control unfed group to 1,060 lmol/l t 8 h post-feeding; J mm (μmol/g/h) Fed Unfed high externl mmoni Control no HEA Fig. 3 Effects of 6-h HEA exposure on mmoni excretion rtes (J mm ) in oth fed nd unfed fish. Feeding occurred t 0 h; HEA exposure egn t 4 h. Control groups represent the verge of 6-h unfed nd fed, no HEA experiments. Crosses represent vlues tht re significntly different from the unfed control; doule crosses represent those tht differ from the fed control. Asterisk indictes significnt difference etween the two controls. Comprisons etween the controls were mde using Mnn Whitney rnk sum test; comprisons etween controls nd other dt points were mde using n ANOVA on rnks sttisticl nlysis followed y Dunn s post hoc test ginst the control vlues (s comprisons filed normlity test) (P \ 0.05, N = 6 8) T mm (µmol) Fed no HEA Fed HEA Unfed HEA Control unfed Fig. 4 Plsm totl mmoni (T mm ) vlues fter feeding nd during HEA exposure including control vlue from unfed fish. Feeding occurred t 0 h; HEA exposure egn t 4 h. Asterisks represent vlues tht re significntly greter thn the control sed on n ANOVA on rnks sttisticl nlysis followed y Dunn s post hoc test (s comprisons filed normlity test). Mens shring the sme letter re not significntly different from one nother within given time point. Comprisons mong tretments t the sme time points were mde using one-wy ANOVA sttisticl nlysis followed y Fisher LSD post hoc test (P \ 0.05, N = 5 14) this elevtion persisted t 10 h (Fig. 4). When exposed to HEA, unfed fish displyed n immedite threefold increse in plsm T mm (fter 2 h, time corresponding to 6 h in the fed groups). This increse ws followed y susequent smll decline to vlue not significntly different from the plsm T mm of the fish in the fed (no HEA) group t lter time points. When fed fish were exposed to HEA, they showed similr ut somewht greter response (fourfold increse) in plsm T mm in comprison with the unfed HEA group, nd much higher T mm thn in the unfed HEA tretment. Indeed, the T mm responses of these ltter tretments were pproximtely dditive, ccounting for the lrge increses seen in the fed HEA group, t lest t 8 nd 10 h post-feeding (Fig. 4). Sodium uptke nd exchnge In fsted trout, control sodium influx rtes (J N in ) were pproximtely lmol/g/h. Control efflux rtes (J N out ) were slightly greter, so net lnce (J N net ) ws slightly negtive. Feeding induced progressive rise in J N in, which ecme significnt t 6 8 h post-feeding (fourfold increse reltive to the control vlue) such tht (J N net ) ecme positive; J N out ws lso significntly elevted t this time (threefold increse; Fig. 5). However, overll, J N net did not vry significntly throughout the entire experimentl period in this fed, no HEA exposure tretment (Fig. 5). Exposure to HEA (t time corresponding to 4 10 h post-feeding in the fed group) led to n immedite depression in J N in in oth fed nd

7 A Influx Efflux Net Flux unfed fish (t 4 6 h), though this decrese ws not significnt (P = 0.230) in the unfed group (Fig. 5, c). Therefter, J N in tended to recover in oth the fed nd unfed HEA groups despite the persistence of HEA. J N out vlues nd J N net tended to e more negtive during HEA in oth these groups, ut there ws high vriility nd no significnt overll differences from the control (Fig. 5, c). -2 Gill mrna expression J N (μmol/g/h) B C high externl mmoni Control Fig. 5 Effects of feeding, feeding comined with 6-h HEA exposure, nd c exposure to 6 h of HEA lone on unidirectionl (J N in, upwrd light gry rs; J N out, downwrd drk gry rs) nd net rtes (J N net, lck rs) of sodium flux over 10-h time period. The rtes from 6-h control using n unfed group of fish re included. Feeding occurred t time 0; HEA egn t 4 h. Asterisks denote vlues tht re significntly different from their respective control vlues sed on one-wy ANOVA sttisticl nlysis followed y Dunnett s post hoc test ginst the control vlue. In the cse of filed normlity test, n ANOVA on rnks sttisticl nlysis ws used followed y Dunn s method post hoc test (P \ 0.05, n = 8) In ll three tretment groups, significnt increses in the mrna expression levels of trnsporters of interest in the gills were not seen until times corresponding to 6 nd 10 h post-feeding (i.e., 2 nd 6 h of HEA; Fig. 6,, c). There were no significnt chnges t 2 or 4 h. Elevtions in Rhcg2 expression levels were significnt in ll three tretments t times corresponding to 6 nd 10 h post-feeding (i.e., 2 nd 6 h of HEA; Fig. 6). The HEAexposed groups exhiited generlly greter upregultion (fed HEA = 23- nd 93-fold increses; unfed HEA = 111- nd 75-fold increses over the control t 6 nd 10 h, respectively) thn in the fed, no HEA control group (32- nd 30-fold increses over the control t 6 nd 10 h, respectively; Fig. 6). There ws lso sixfold increse in NHE-2 mrna expression t 6 h in fed fish not exposed to HEA; the men vlue t 10 h remined out the sme ut ws no longer significntly different from the control (P = 0.08) ecuse of incresed vriility (Fig. 6). In unfed fish, exposure to HEA cused significnt increse in the expression of NHE-2 t 6 nd 10 h (18- nd 8-fold, respectively), wheres fed fish exposed to HEA displyed tenfold increse only fter 10 h (Fig. 6). The unfed HEA tretment ws the only group to disply significnt increse (sevenfold) in H? ATPse mrna expression t 6 h; however, the increse in the fed only tretment ws close to eing significnt (P = 0.07; Fig. 6c). After 10 h, ll tretment groups showed significnt increses in H? ATPse expression (10-, 11-, nd 10-fold for fed no HEA, fed HEA, nd unfed HEA groups, respectively; Fig. 6c). Brnchil H? -ATPse ctivity Brnchil H? -ATPse ctivity incresed significntly y out 1.7-fold t 4 h fter the mel in the fed fish (Fig. 7). Therefter, ctivity tended to decline, with tendency for greter decreses in the two HEA tretments. However t 6 nd 10 h, none of these vlues were significntly different from the control or from one nother.

8 A Fed no HEA Fed HEA Unfed HEA Control Rhcg2 Activity (μmol ADP/mg protein/h) Fed no HEA Fed HEA Unfed HEA mrna Expression B C Col 10 Col 16 Rh Col 22 NHE-2 H + -ATPse 0.0 Control Fig. 7 H? -ATPse ctivity in the gills of unexposed (no HEA) fed fish s well s fed nd unfed fish exposed to HEA. The control vlue represents gene expression in the gills of unfed, no HEA fish. Smpling for oth fed groups (HEA nd non-hea) took plce t 2, 4, 6, 8, nd 10 h. The 2 nd 4 h time points (where fish re yet to e exposed to HEA) re the sme for ech of these groups, s the tretment up to these times ws identicl, nd so only single vlue is shown t ech of these time points. Smpling for the unfed HEA group took plce only t 6, 8, nd 10 h, s 2 nd 4 h points for this tretment would represent the unfed, no HEA control. Feeding occurred t 0 h; HEA exposure egn t 4 h. Asterisks denote vlues tht re significntly different from the control levels sed on n ANOVA on rnks sttisticl nlysis followed y Dunn s post hoc test (s ll comprisons filed normlity test). Mens shring the sme letter re not significntly different from one nother within given time point (P \ 0.05, N = 5 6) Control Fig. 6 mrna expression of Rhcg2, NHE-2, nd c H? -ATPse reltive to totl RNA in the gills of unexposed (no HEA) fed fish s well s fed nd unfed fish exposed to HEA. The control vlue represents gene expression in the gills of unfed, no HEA fish. Smpling for oth fed groups (HEA nd non-hea) took plce t 2, 4, 6, 8, nd 10 h. The 2 nd 4 h time points (where fish re yet to e exposed to HEA) re the sme for ech of these groups, s the tretment up to these times ws identicl, nd so only single vlue is shown t ech of these time points. Smpling for the unfed HEA group took plce only t 6, 8, nd 10 h, s 2 nd 4 h points for this tretment would represent the unfed, no HEA control. Feeding occurred t 0 h; HEA exposure egn t 4 h. Asterisks denote vlues tht re significntly different from the control levels sed on n ANOVA on rnks sttisticl nlysis followed y Dunn s post hoc test (s ll comprisons filed normlity test). Mens shring the sme letter re not significntly different from one nother within given time point. Since ll comprisons mong tretments t given time point filed normlity test, ll comprisons were mde using n ANOVA on rnks sttisticl nlysis followed y Dunn s post hoc test (P \ 0.05, N = 5 6) Discussion Overview Two of our originl three hypotheses were confirmed. Firstly, feeding did cuse upregultion of Rhcg2, NHE-2, nd H? -ATPse mrna expression in the gills, trnsient increse in rnchil H? -ATPse ctivity, nd trnsient rise in N? influx. Though the mrna expression dt do not explicitly demonstrte tht the expression or ctivity of the ssocited proteins incresed, they certinly suggest role for these proteins during feeding nd in response to HEA s demonstrted in other tretments from this study. Notly, for one gene (H? -ATPse), we recorded oth mrna expression nd functionl ctivity nd found mrked temporl disconnect etween the two. Secondly, fter feeding, trout were le to excrete mmoni ginst HEA more effectively thn unfed fish. With respect to our third hypothesis, the results re complex. HEA did inhiit N? influx s predicted, though the effect ws significnt only in the fed fish. Ammoni excretion ws not inhiited t ll y HEA in fed fish, so in sense recovery of mmoni excretion in the fce of HEA ws more rpid in fed fish. However, contrry to prediction, there ws no

9 difference in the rtes t which N? influx recovered in the fce of continued HEA etween fed nd unfed fish. As outlined elow, we elieve the interprettion of these results my e more complicted thn the originl ides ehind our hypotheses, involving importnt roles for simple diffusion, for chnging P NH3 grdients, for non-genomic modifiction of trnsporter ctivity, nd for uncoupling of N? uptke from H? nd mmoni efflux during HEA. To explore the role of P NH3 grdients from lood plsm to wter, we hve estimted them sed on individul mesurements of ulk wter T mm, plsm T mm, wter ph, nd n ssumed constnt lood plsm ph of 7.9 (Fig. 7). Becuse of the smll size of the fish, it ws not possile to otin ccurte mesurements of plsm ph y cnnultion, nd mesurements of plsm T mm my hve een elevted y the disturnce of cudl severnce smpling (discussed y Wood 1993). Furthermore, without knowledge in these smll fish of the existence or time course of the postprndil lkline tide, which occurs in dult trout (Cooper nd Wilson 2008; Bucking nd Wood 2008), this fctor, which would potentilly elevte plsm P NH3,could lso not e tken into ccount. Bulk wter ph mesurements overlook possile cidifiction of the micro-environment in the gill oundry lyer. Therefore, the oserved P NH3 grdients should e tken s indictive of generl trends only (Fig. 8). Also importnt to note is tht T mm grdients (which my lso e indictive of mmoni excretion grdients s Rh mmoni trnsport processes hve not yet een fully resolved) showed very similr trends nd thus hve not een shown. Nevertheless, these dt re sufficient to indicte tht the ulk P NH3 grdient ws positive (i.e., in fvor of excretion) throughout most of the post-feeding period in fed fish, which were not exposed to HEA. In contrst, the grdient ws highly negtive throughout the period of HEA in oth unfed nd fed tretments, indeed somewht more so in the ltter, so tht these fish were chieving mmoni excretion ginst ulk P NH3 grdients. Effects of feeding lone In previous studies on freshwter trout, feeding hs een demonstrted to cuse increses in oth J mm nd plsm T mm (Kushik nd Teles 1985; Wicks nd Rndll 2002, ; Wood 1993; Gelineu et l. 1998; Bucking nd Wood 2008). In the present study, J mm incresed significntly s erly s 2 4 h post-feeding (Fig. 1), ut significnt increses in plsm T mm did not occur until 8 h (Fig. 4). These oservtions differ from those of Bucking nd Wood (2008) on dult trout ( g) held in wter of identicl chemistry, where increses in plsm T mm occurred within 2 4 h postfeeding nd preceded increses in J mm. Bucking nd Wood (2008) lso showed the sl level of plsm T mm to e pproximtely 100 lm, wheres levels in this study were Δ P NH3 (μtorr) Fed no HEA Fed HEA Unfed HEA Control unfed Fig. 8 Effects of feeding, HEA exposure or oth feeding nd HEA exposure on the estimted prtil pressure grdient of NH 3 from lood to wter. Feeding occurred t 0 h; HEA egn t 4 h. Asterisks represent vlues tht re significntly different from their respective control vlues sed on one-wy ANOVA sttisticl nlysis followed y Dunnett s post hoc test (or, in the cse of filed normlity test, n ANOVA on rnks sttisticl nlysis followed y Dunn s method post hoc test ws used). Mens shring the sme letter re not significntly different from one nother within given time point. Comprisons mong tretments t given time point were mde using one-wy ANOVA sttisticl nlysis followed y Fisher LSD post hoc test. In the cse of filed normlity test, n ANOVA on rnks sttisticl nlysis ws used followed y Dunn s method post hoc test (P \ 0.05, N = 5 14) pproximtely five times tht (Fig. 4). It is possile tht this difference ws due to smpling techniques (cudl puncture vs. til severing method), stress effects, or differing metolisms of the vrying sizes of fish. However, for the purposes of this study, we re more concerned in the qulittive nd temporl trends, thn in the solute vlues. The smller fish (3 9 g) used in the present study therefore pper to upregulte excretion in response to feeding more quickly thn do lrger trout. We interpret the lck of detectle chnge in plsm T mm over the first 6 h post-feeding to men tht J mm is incresed sufficiently y 2 4 h to mintin norml plsm T mm levels for some time. To our knowledge, the present study is the first to demonstrte tht gill Rhcg2, NHE-2, H? -ATPse expression levels, s well s rnchil H? -ATPse ctivity levels, re incresed y feeding. The ltter response ws significnt t 4 h post-feeding, while the Rhcg2 nd NHE-2 mrna expression increses were significnt t 6 h, wheres the H? -ATPse mrna expression increse did not ecome significnt until 10 h. These results provide cutionry note s to the importnce of strict control of the feeding regime in ny future studies on gill trnsporter expression nd ctivity. Interestingly, Rhcg2 expression lso incresed fter mel in dult rinow trout (C.M. Nwt nd C.M. Wood, unpulished results), ut not until 12 h post-feeding, re-enforcing the ide tht smll trout respond more quickly. c c

10 We interpret these s responses to nturl internl mmoni loding ssocited with de-mintion of sored mino cids nd proteins, comprle to those seen when trout were infused with exogenous mmonium slts (Nwt nd Wood 2009), though it is possile tht the complex hormonl events ssocited with feeding (Buddington nd Krogdhl 2004) my hve plyed modulting role. The elevtion of J mm y 2 4 h post-feeding (Fig. 1) nd the mintennce of norml levels of plsm T mm through 6 h (Fig. 4) suggest tht the rnchil mmoni excretion mechnism(s) were quickly upregulted fter feeding. This is not ovious from the mrna expression of the puttive trnsport proteins, since t 2 nd 4 h post-feeding there were no significnt increses in Rhcg2, NHE-2, or H? -ATPse (Fig. 6). However, these dt represent only the messges for the proteins, nd it ws instructive to find tht rnchil H? -ATPse ctivity hd incresed significntly y 4 h postfeeding, long efore the significnt increse in mrna expression t 10 h. Indeed, it is very possile tht nongenomic upregultion of the ctivity of existing proteins ws sufficient for erly elevtion of J mm, prior to lter, lrger increses in J mm, which my hve een due to the significntly incresed plsm T mm levels occurring t 8 h (Figs. 1, 4) ssocited with slow processing of the dietry N-lod. In fct, Seshdri et l. (2006) demonstrted tht when cidosis is induced in rts, condition tht is known to cuse n increse in urinry mmoni excretion, Rhcg protein expression incresed in severl res of the kidney though mrna expression of Rhcg remined unchnged. In follow-up study, it ws demonstrted tht cidosis induced n increse in picl protein expression of Rhcg nd decrese in cytosolic protein expression, suggesting tht these proteins re present in vesicles nd re inserted during cidosis (Seshdri et l. 2006). This, together with the erly trnsient increse in H? -ATPse ctivity t 4 h, is potentil explntion for the increse in mmoni excretion cpcity, which occurs in the sence of n increse in mrna expression demonstrted here. The delyed nd lrger increse in J mm seen 8 h fter mel (Fig. 1) ws presumly ssocited with increses in protein synthesis ssocited with increses in Rhcg2 nd NHE-2mRNA levels t 6 h post-feeding (Fig. 6, ). It ws not until 10 h postfeeding tht significnt increse in H? -ATPse mrna lso occurred (Fig. 6c), perhps response to the increse in plsm T mm tht occurred t 8 h (Fig. 4). A generlly higher ulk P NH3 grdient fvoring excretion ws seen throughout most of the post-feeding period (Fig. 8) nd my lso hve contriuted to the sustined elevtion in J mm,y either Rh-medited trnsport (Nwt et l. 2010) or simple diffusion (Kelly nd Wood 2001; Tsui et l. 2009). If postprndil lkline tide (Bucking nd Wood 2008; Cooper nd Wilson 2008) occurs in these juvenile trout, then the contriution of this component would e incresed. Also criticl to the induction of elevted J mm ws the oserved trnsient increse in J N in, which ecme significnt t 6 h fter the mel (Fig. 6). This trnsient increse, which my e relted to sustined increse in J mm, could e n indiction of the rther loose coupling etween these processes. In situ studies of MRCs in the lrve of Jpnese medk indicted tht mmoni excretion through Rhcg (Rhcg1) ws dependent on H? excretion y NHE-3, which ppered to e tightly linked to N? uptke (Wu et l. 2010), These were similr, ut not identicl to the findings of Tsui et l. (2009) where in vitro studies on cultured trout gill epitheli indicted tht Rhcg-2, NHE-2, nd H? - ATPse ll plyed role in the linkge of portion of mmoni excretion to N? uptke, wheres portion occurred y simple diffusion long P NH3 grdients. In vivo, this increse in J N in proly occurred in response to internl mmoni loding ssocited with feeding, since the infusion of exogenous mmonium slts is lso known to cuse n increse in J N in in intct fish (Kerstetter nd Keeler 1976; Metz nd Grci-Romeu 1964; Metz 1973; Kerstetter nd Keeler 1976; Wilson et l. 1994; Slm et l. 1999). The increse in rnchil H? -ATPse ctivity seen t 4 h post-feeding my lso hve plyed role here. To our knowledge, this is the first demonstrtion tht feeding norml mel cuses n increse in J N in in freshwter fish; notly, J N out lso incresed, so N? homeostsis ws preserved (Fig. 6). Previous studies hve insted focused on the inhiition of J N in nd lrge elevtions of J N out, which re ssocited with ingestion of experimentl high- NCl mels (Smith et l. 1995; Pyleetl. 2003). Overll, these oservtions nd the increses in mrna expression of NHE-2 (Fig. 6) nd H? ATPse (Fig. 6c) demonstrte tht the hndling of mmoni post-feeding is likely to e, t lest in prt, coupled to N? uptke. Thus, on n overll sis, feeding cuses similr responses in gill mrna expression ptterns, J N in, nd J mm to mmonium slt infusion (Wilson et l. 1994; Slm et l. 1999; Nwt nd Wood 2009). These in vivo results support the N?? /NH 4 exchnge complex model of Rhcg2, NHE-2, H? ATPse function proposed y Wright nd Wood (2009), which loosely links Rhcg2-medited NH 3 excretion to ctive N? uptke through oundry lyer cidifiction. As originlly proposed y Heisler (1990), there my e plsm T mm threshold ove which this ctive mechnism cuts in. Effects of HEA exposure in unfed fish Exposure to HEA led to n initil reversl of mmoni excretion (Figs. 2, 3) s erlier demonstrted y Wilson et l. (1994) nd Nwt et l. (2007) in unfed dult trout. In response to HEA exposure, there ws drmtic increse in plsm T mm, which ws rpid (within 2 h), compred to the delyed nd less prominent increse seen

11 in response to feeding (within 8 h; Fig. 4). This increse occurred in conjunction with the reversl of mmoni excretion during HEA (Figs. 2, 3). A trend of decresing plsm T mm ensued (Fig. 4) s unfed fish were le to reestlish mmoni excretion y 4 6 h of exposure to HEA (Fig. 3). This ility to overcome lrge inwrdly directed P NH3 grdient (Fig. 7) my e ttriuted to the induction of rnchil mmoni excretion mechnisms, which re potentilly ctive. Exposure to HEA led to n increse in the mrna expressions of Rhcg2, NHE-2, nd H? -ATPse mrna fter only 2 h of exposure (corresponding to the 6 h time point in Fig. 6), similr to responses to mmonium slt infusion (Nwt nd Wood 2009). These increses were more immedite thn the increses seen in response to feeding, which did not occur until 6 10 h fter the mel (Fig. 6). Notly, these responses to HEA occurred more rpidly thn in dult trout fced with the sme HEA chllenge in identicl wter chemistry (Nwt et l. 2007). In the dult trout, similr increses in Rhcg2 nd H? - ATPse mrna expression were not seen until h, nd re-estlishment of positive J mm did not occur until h (Nwt et l. 2007), gin highlighting the more rpid responses of these juvenile trout. High environmentl mmoni exposure tended to depress J N in, though not y significnt mrgin (Fig. 5c). This response contrsts with the response to feeding lone, where J N in (likely coupled to oundry lyer cidifiction) ws clerly incresed (Fig. 5) s puttive mechnism enhncing J mm (Fig. 1). This inhiition of J N in y HEA is similr to results reported y Twitchen nd Eddy (1994) nd Wilson et l. (1994) nd my e ttriuted to competition y NH? 4 with N? for externl inding sites on N? trnsport proteins (N? chnnels nd/or NHE-2). Whether this simply slows down N? uptke, or whether it results in greter NH? 4 uptke (i.e., incresed mmoni loding) in sustitution for N? uptke through these pthwys is unknown. However, the ltter would clerly e mldptive, otherwise why then upregulte NHE-2 nd H? -ATPse mrna during HEA exposure (Fig. 6, c)? A more likely scenrio is tht N? influx is slowed, ut H? efflux is mintined or incresed. This scenrio envisges tht N? uptke my e uncoupled from H? flux nd mmoni excretion. Using low doses of miloride (10-5 M) in the rown trout, Slmo trutt, Nelson et l. (1997)demonstrted tht J N in could e reduced y 75% while H? efflux nd J mm continued t control rtes. We speculte tht during HEA exposure, NH? 4 cts like low dose miloride, such tht H? efflux nd oundry lyer cidifiction for trpping of Rhcg2-medited NH 3 efflux cn still occur. By this scenrio, upregultion of NHE-2 would serve to mintin some degree of N? uptke nd linked H? excretion, while H? - ATPse (now uncoupled from N? uptke), together with CO 2 hydrtion, would provide the ulk of the H? efflux nd cid trpping mechnism. H? -ATPse mrna ws upregulted (Fig. 6c), wheres H? -ATPse ctivity remined t control levels though with non-significnt tendency to decline in HEA (Fig. 7). Presumly, H? -ATPse ctivity would increse over the longer term if HEA were mintined, s reported y Nwt et l. (2007) in dult trout. Protective effects of feeding ginst HEA exposure Unlike unfed fish exposed to HEA, fed fish were le to immeditely mintin norml mmoni excretion rtes when exposed to HEA, demonstrting tht feeding hs protective effect ginst HEA exposure (Fig. 3). There ws no initil reversl of J mm to negtive vlues. These fed trout lso displyed increses in plsm T mm during HEA similr to or greter thn those seen in the unfed, HEA group, reflecting the dditive effect of feeding (Fig. 4). However, on n individul fish sis, the ulk P NH3 grdient ginst which they were excreting ws in fct even more negtive thn in the unfed HEA group (Fig. 7), owing to the fct tht they were ctively excreting mmoni into the wter from the eginning of the exposure, effectively incresing the P NH3 grdient y incresing wter T mm. Contrry to our originl ides (see Introduction ), this incresed ility to excrete mmoni ginst the grdient cnnot e ttriuted to prior upregultion of Rhcg2, NHE-2, nd H? -ATPse mrna expression ssocited with feeding, for two resons. Firstly, fter 2 h of HEA (6 h time point in Fig. 6), upregultion of messge in these fed fish exposed to HEA ws quntittively equl to or less thn in fed fish, which hd not een exposed to HEA t the sme time. Secondly, compred to unfed fish exposed to HEA, fed fish exposed to HEA demonstrted delyed upregultion of mrna expression of NHE-2 nd H? -ATPse, which ws never greter in mgnitude (Fig. 6, c). Though the upregultion of Rhcg2 mrna expression ws not delyed, it lso ws not greter in mgnitude thn tht seen in unfed fish exposed to HEA (Fig. 6). We speculte tht the explntion for this pprent prdox lies in the upregulted mmoni excretion cpcity seen erly on in response to feeding lone (within 2 4 h fter the mel), without mrna recruitment, s discussed erlier. It is likely tht y the time of HEA exposure (4 h postfeeding), fed fish lredy possessed incresed mmoni trnsport ctivity levels in their gill cells due to non-genomic modifiction. Note for exmple the significnt increse in rnchil H? -ATPse ctivity t 4 h post-feeding (Fig. 7), prior to ny significnt increse in H? -ATPse messge (Fig. 6c). Any lter protein synthesis ssocited with mrna increses would likely dd to this ctivity. This my e the explntion for the finding of Wicks nd Rndll (2002) tht feeding protects rinow trout from mmoni toxicity, elevting the LC50 during the first 24 h of HEA exposure.

12 Unlike the unfed HEA group, fed fish exposed to HEA displyed depression of J in N, which ws significnt (Fig. 5), nd recovery of J in N ws certinly no fster even though J mm ws well mintined (Fig. 3). It is uncler why the inhiitory effect of high NH 4? should e greter in fed fish; perhps, these trout were relying more on NHE-2 t this time compred to unfed fish. Their rnchil H? - ATPse ctivity levels lso tended to e lower (Fig. 7). Regrdless, these dt demonstrte tht upregultion of N? influx is not requirement for ctive mmoni excretion ginst the P NH3 grdient during HEA exposure. The depression of J in N my seem counter-intuitive given tht expressions of oth NHE-2 (Fig. 6) nd H? -ATPse mrna (Fig. 6c) were incresed significntly t 6 h fter exposure to HEA (10 h fter the mel). However, s in the cse of the unfed HEA group, it is likely tht NH 4? ws competing for N? inding sites, such tht H? efflux nd mmoni excretion were then uncoupled from N? uptke t tht time. Perhps with longer HEA exposure, more complete recovery or increse of J in N would hve een seen in oth HEA tretments. Conclusions Rhcg2, NHE-2, nd H? -ATPse ll pper to ply significnt roles in mmoni excretion in trout in vivo, in ccordnce with the N? /NH? 4 exchnge complex model of Wright nd Wood (2009), ut simple diffusive flux long the P NH3 grdient is lso importnt. Juvenile trout cn elevte mmoni excretion more rpidly thn dult trout in response to feeding or HEA. After feeding, the mmoni excretion cpcity of the gills increses efore elevted Rhcg2, NHE-2, nd H? -ATPse mrna levels re seen, suggesting non-genomic ctivtion of existing trnsport protein function. This confers greter cpcity for fed fish to excrete mmoni ginst ulk P NH3 grdient in the fce of HEA. Lter fter feeding, elevted N? influx ppers to ply n importnt role in ugmenting mmoni excretion, ut this is not the cse during HEA exposure. The influx of N? is loosely tied to n efflux of H? (either y NHE-2 or H? -ATPse) nd it is this efflux of H?, not the influx of N?, which is responsile for fcilitting mmoni excretion. This ecomes evident during HEA where N? influx is depressed even though Rhcg2, NHE-2, nd H? -ATPse re ll upregulted nd mmoni excretion is restored: i.e., N? influx is uncoupled from H? nd mmoni efflux. In future studies, it would e interesting to exmine mmoni nd net H? excretion during HEA when N? is not present in the wter. This would quntify whether or not mmoni excretion during HEA (in fed or unfed fish) cn, in fct, e completely uncoupled from N? influx. Studies exmining the post-trnscriptionl regultion of the implicted trnsporters will lso e essentil to relizing the complete model for mmoni excretion during feeding nd HEA exposure. The temporl disconnect etween recorded chnges in H? -ATPse mrna expression nd functionl ctivity oserved in the present study underscores the need for such studies. Finlly, the use of phrmcologicl lockers to inhiit H? excretion (filomycin, EIPA, nd cetzolmide) during HEA would e useful in determining the reltive contriution to mmoni excretion of H? - ATPse, NHE-2, nd cronic nhydrse, respectively. Acknowledgments This study ws funded y n NSERC Discovery Grnt to CMW, who is lso supported y the Cnd Reserch Chir Progrm. The experiments were pproved y the institutionl niml cre committee t McMster University. References Avell M, Bornncin M (1989) A new nlysis of mmoni nd sodium trnsport through the gills of the freshwter rinow trout (Slmo girdneri). J Exp Biol 142: Bkouh N, Benjelloun F, Hulin P, Brouillrd F, Edelmn A, Cherif-? Zhr B, Plnelles G (2004) NH 3 is involved in the NH 4 trnsport induced y the functionl expression of the humn Rh C Glycoprotein. J Biol Chem 279: Bucking C, Wood CM (2008) The lkline tide nd mmoni excretion fter voluntry feeding in freshwter rinow trout. J Exp Biol 211: Buddington RK, Krogdhl A (2004) Hormonl regultion of the fish gstrointestinl trct. Comp Biochem Physiol A 139: Bustin SA (2000) Asolute quntifiction of mrna using rel-time reverse trnscription polymerse chin rection ssys. J Mol Endocrinol 25: Bustin SA (2002) Quntifiction of mrna using rel-time reverse trnscription PCR (RT-PCR): trends nd prolems. J Mol Endocrinol 29:23 39 Cmeron JN, Heisler N (1983) Studies of mmoni in the rinow trout: physico-chemicl prmeters, cid se ehviour, nd respirtory clernce. J Exp Biol 105: Cooper CA, Wilson RW (2008) Post-prndil lkline tide in freshwter rinow trout: effects of mel nticiption on recovery from cid se nd ion regultory disturnces. J Exp Biol 211: Fromm PO (1963) Studies on renl nd extr-renl excretion in freshwter teleost, Slmo girdneri. Comp Biochem Physiol 10: Fromm PO, Gillette JR (1968) Effect of mient mmoni on lood mmoni nd nitrogen excretion of rinow trout (Slmo girdneri). Comp Biochem Physiol 26: Gelineu A, Medle F, Boujrd T (1998) Effect of feeding time on postprndil nitrogen excretion nd energy expenditure in rinow trout. J Fish Biol 52: Heisler N (1990) Mechnisms of mmoni elimintion in fishes. In: Truchot JP, Lhlou B (eds) Animl nutrition nd trnsport processes. Trnsport, respirtion nd excretion: comprtive nd environmentl spects. Comp Physiol, vol 2, Krger, Bsel, pp Hung CYC, Tsui KNT, Wilson JM, Nwt CM, Wood CM, Wright PA (2007) Rhesus glycoprotein gene expression in the mngrove killfish Kryptoleis mrmortus exposed to elevted environmentl mmoni levels nd ir. J Exp Biol 210:

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