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1 623 The Journl of Experimentl iology 216, Pulished y The Compny of iologists Ltd doi:1.1242/je.7441 RESERCH RTICLE Mechnisms of trnsepithelil mmoni excretion nd luminl lkliniztion in the gut of n intestinl ir-rething fish, Misgurnus nguillicudtus Jonthn M. Wilson 1, *, Jon Moreir-Silv 1,2, Inês L. S. Delgdo 1, Sue C. Enks 3, Mthilkth M. Vijyn 4, João Coimr 1,2 nd Mrtin Grosell 3 1 Lortório de Ecofisiologi, CIMR/CIIMR Centro Interdisciplinr de Investigção Mrinh e mientl Universidde do Porto, Portugl, 2 Instituto de Ciêncis iomédics de el Slzr d Universidde do Porto, Portugl, 3 Rosenstiel School of Mrine nd tmospheric Science/Division of Mrine iology nd Fisheries, University of Mimi, Mimi, US nd 4 Deprtment of iology, University of Wterloo, Wterloo, ON, Cnd *uthor for correspondence (wilson.jm.cimr@gmil.com) SUMMRY The wetherloch, Misgurnus ngullicudtus, is n intestinl ir-rething, freshwter fish tht hs the unique ility to excrete mmoni through gut voltiliztion when rnchil nd cutneous routes re compromised during high environmentl mmoni or ir exposure. We hypothesized tht trnsepithelil gut NH + 4 trnsport is fcilitted y n picl N + /H + (NH + 4 ) exchnger (NHE) nd solterl N + /K + (NH + 4 )-TPse, nd tht gut oundry lyer lkliniztion (NH + 4 NH 3 + H + ) is fcilitted y picl HCO 3 secretion through Cl /HCO 3 nion exchnger. This ws tested using phrmcologicl pproch with nterior (digestive) nd posterior (respirtory) intestine preprtions mounted in ph-stt-equipped Ussing chmers. The nterior intestine hd mrkedly higher conductnce, incresed short-circuit current, nd greter net se (J se ) nd mmoni excretion rtes (J mm ) thn the posterior intestine. In the nterior intestine, HCO 3 ccounted for 7% of J se. In the presence of n imposed serosl mucosl mmoni grdient, inhiitors of oth NHE (EIP,.1 mmol l 1 ) nd N + /K + -TPse (ouin,.1 mmol l 1 ) significntly inhiited J mm in the nterior intestine, lthough only EIP hd n effect in the posterior intestine. In ddition, the nion exchnge inhiitor DIDS significntly reduced J se in the nterior intestine lthough only t high dose (1 mmol l 1 ). Cronic nhydrse does not pper to e ssocited with gut lkliniztion under these conditions s ethoxzolmide ws without effect on J se. Memrne fluidity of the posterior intestine ws low, suggesting low permeility, which ws lso reflected in lower mucosl serosl J mm in the presence of n imposed grdient, in contrst to tht in the nterior intestine. To conclude, lthough the posterior intestine is highly modified for gs exchnge, it is the nterior intestine tht is the likely site of mmoni excretion nd lkliniztion leding to mmoni voltiliztion in the gut. Supplementry mteril ville online t Key words: mmoni voltiliztion, chloride icronte exchnger, sodium proton exchnger, sodium potssium TPse, mmonium. Received 26 pril 212; ccepted 8 Octoer 212 INTRODUCTION The wetherloch (Misgurnus nguillicudtus) is non-oligte intestinl ir-rething freshwter fish (Grhm, 1997; McMhon nd urggren, 1987). The gstrointestinl trct of the loch is highly modified for gs exchnge, with the posterior intestine (two-thirds of the totl gut length) lcking chrcteristic sorptive columnr enterocytes, nd insted hving well-vsculrized strtified epithelium with intrepithelil cpillries suitle for gs exchnge (McMhon nd urggren, 1987; Gonçlves et l., 27; Wilson nd Cstro, 21). The loch ccomplishes intestinl ir-rething y swllowing ir nd pssing it down the length of the gut unidirectionlly. The intestine is chrcteristiclly inflted with ir even during feeding, with the intestinl fluid limited to surfce film (Jeuken, 1957; McMhon nd urggren, 1987). Tsui nd collegues (Tsui et l., 22) provided evidence tht the gut is the site of mmoni voltiliztion, the rte of which increses from.5 to.8 mmol totl mmoni nitrogen (TN) g 1 dy 1 when rnchil nd cutneous routes of mmoni excretion re compromised (>9%) y ir exposure or high environmentl mmoni (HE) levels. The use of the gut s n ccessory ir-rething orgn for oth oxygen uptke nd mmoni excretion llows the loch to survive periods of drought nd poor wter qulity (hypoxi, HE) tht chrcterize its nturl environment. Consequently, the wetherloch is very mmoni-tolernt fish (Chew et l., 21; Tsui et l., 22; Moreir-Silv et l., 21). Under mmoni-loding conditions (HE or emersion), the ccumultion of mmoni in the ody together with the increse in lood ph will fvor n outwrd grdient of mmoni (Tsui et l., 22). This evidence would rgue in fvor of pssive trnsepithelil NH 3 diffusion s the prole mechnism for mmoni excretion; however, lkliniztion of the gut luminl surfce from ph 7.4 up to ph 8.2 hs lso een oserved (Tsui et l., 22), which would e inconsistent with this mechnism s ckflux of NH 3 would occur. Insted, we propose fcilitted trnsport of NH 4 + s the mechnism of mmoni excretion. NH 4 + hs een shown to e trnsported y N + /H + exchngers (NHEs) nd N + /K + -TPse y replcing H + nd K +, respectively (Knepper et l., 1989; Mllery, 1983; Rndll et l., 1999). oth these trnsporters hve een well chrcterized in the verterte intestine, with the solterl N + /K + -TPse hving n importnt THE JOURNL OF EXPERIMENTL IOLOGY

2 624 The Journl of Experimentl iology 216 (4) role in driving trnsepithelil trnsport processes (nutrient nd ion regultion) (Grosell, 21; Mrshll nd Grosell, 26). In mmmls, the intestinl NHEs re involved in intrcellulr ph regultion nd N + uptke (Zchos et l., 25). The NHE isoform 1 (NHE1) is expressed t the solterl memrne, wheres NHE2 is picl nd NHE3 is recycled etween intrcellulr vesicles nd the plsm memrne (Zchos et l., 25). Gut lumen surfce lkliniztion in the loch is proly ccomplished through HCO 3 excretion in exchnge for Cl s hs een reported in mrine fishes (Grosell et l., 29). lthough intestinl se excretion hs een reported in freshwter trout (Genz et l., 211), the mechnism of luminl lklinizton hs only een well chrcterized in mrine fishes, which imie wter for osmoregultion (Grosell, 26). The intestinl nion exchnger responsile for lkliniztion in mrine teleosts hs een determined to e Slc266 (Kurit et l., 28; Grosell et l., 29) lthough in freshwter trout gut expression is generlly sent (Genz et l., 211). In the present study, the mechnisms of mmoni nd se excretion in the wetherloch gut were chrcterized using phrmcologicl pproch. Investigtion of the roles of N + /K + - TPse nd NHE on NH 4 + trnsport utilized the inhiitors ouin nd EIP (see elow), respectively, while the roles of the Cl /HCO 3 nion exchnger nd cronic nhydrse (C) in gut lkliniztion were ssessed using DIDS (see elow) nd ethoxzolmide, respectively. The electrophysiologicl properties of the different regions of the gut (nterior versus posterior intestine) nd their memrne fluidity were lso ssessed. MTERILS ND METHODS nimls dult wetherloches, M. nguillicudtus (Cntor 1842), were purchsed from the min wet mrket in Yuen Long, Hong Kong, nd mintined t the Deprtment of iology nd Chemistry, City University, Hong Kong. Fish for Ussing chmer experiments were trnsported to the Rosenstiel School of Mrine nd tmospheric Science, University of Mimi, y ir freight with miniml wter. Upon rrivl, fish were mintined in 1 l flow-through glss quri contining dechlorinted Virgin Key tp wter (N +.35 mmol l 1, hrdness 45 mg l 1 CCO 3, ph 7.5), t 25 C, under nturl light conditions. During this period the fish were fed dily d liitum with commercil fish food (Grnured, Ser GmH, Germny), nd the feed ws withheld 48 h prior to the strt of experimentl procedures. Procedures were pproved y the University of Mimi niml Cre nd Use Committee. In vitro mmoni flux experiment Wetherloches (1.97±6.68 g nd 11.2±1.6 cm, mens ± s.d.) were treted with n overdose of neutrlized tricne methnesulfonte [1:5 (w/v), Syndel L, Qulicum ech, C, Cnd] nd killed y decpittion. The entire gut ws excised, cut ventrlly long its entire length nd lid out over pper towel moistened with rtificil serosl sline (Tle 1), for immedite mounting in the Ussing chmer system (P23, Physiologic Instruments, Sn Diego, C, US). Electrophysiology nd ph-stt nlysis The nterior nd the most posterior region of the intestine were mounted on tissue-holding cssettes [P243 (.1 cm 2 ) to P2413 (.71 cm 2 ), depending on the size of the tissue; Physiologic Instruments]. The tissue inserts were mounted in Ussing chmers nd oth serosl nd mucosl chmers were filled with 1.6 ml of pre-gssed serosl sline (see Tle 1 for sline composition) Tle 1. Composition nd properties of modified Cortlnd slines (Wolf, 1963) used in the Ussing chmer experiments Serosl (mmol l 1 ) Mucosl (mmol l 1 ) NCl KCl CCl MgSO NH 2 PO 4 3. N 2 HPO 4 3. NHCO Glucose.1% Mnnitol 1 Gs.3% CO 2 in O 2 O 2 ph Osmollity The mucosl sline ws titrted to ph 7.8 with NOH. mintined t 25 C. The sline solutions were continully gssed with.3% CO 2 /O 2, to ensure the proper mixing of oth hlfchmers. Current nd voltge electrodes were connected to n mplifier (VCC6, Physiologic Instruments), nd dt trnsferred to PC using the IOPC systems interfce hrdwre nd cqknowledge softwre (version 3.8.1). Electrophysiologicl mesurements were performed under symmetric conditions (see Grosell nd Genz, 26) using voltge clmp ( mv) with mucosl reference while current ws recorded. 5 mv potentil ws induced for 3 s, t 6 s intervls, through the epithelium from the mucosl to the serosl side. Current nd voltge dt otined together with the re of the exposed tissue permitted the clcultion of epithelil conductnce s follows: G = I / (V ), (1) where G is conductnce (μs cm 2 ), I is current, V is electricl potentil nd is tissue re. The sline in the mucosl chmer ws then removed nd the chmer rinsed nd refilled with mucosl sline (1.6 ml), nd gssed with O 2 for stle titrtions. During the course of the flux experiments descried elow, G nd trnsepithelil potentil (TEP) were mesured under current-clmp conditions with 5 μ pulses (3 s) every 6 s. For se nd mmoni flux mesurements, the preprtions were mintined under symmetric, current-clmp conditions in the sence of ph nd osmotic grdients. The ph electrode nd microurette of the ph-stt system were inserted into the mucosl chmer to mesure the net se flux using ph-stt technique (Grosell nd Genz, 26). In rief, the mucosl slines were mintined t 7.8±.3 ph units y the ddition of.5 mol l 1 HCl, s mesured with comintion ph electrodes (PHC4.8, Rdiometer, Copenhgen, Denmrk) nd titrted with utomicrourettes, oth connected to ph-stt titrtion systems (TIM 854 or 856, Rdiometer). The titrtion dt were trnsferred to PC using Titrmster softwre (versions 1.3 nd 2.1) for further nlysis, nd se secretion rte (J se ) ws clculted s: J se = H + / ( t), (2) where H + is the mount of.5 mol l 1 HCl dded y the ph-stt system over time (t) in 1 h intervls per re () of tissue insert (cm 2 ). Net se flux is expressed s μequiv cm 2 h 1. Positive vlues indicte net se flux into the mucosl comprtment. n initil 1 h control flux with stle TEP nd se secretion rtes ws followed y 2 h experimentl flux period with 1 mmol l 1 NH 4 Cl on the serosl side dded s 1 stock solution, nd finlly THE JOURNL OF EXPERIMENTL IOLOGY

3 Gut mmoni excretion nd lkliniztion mechnisms in loch 625 y 2 h experimentl flux with 1 mmol l 1 NH 4 Cl on the serosl side nd phrmcologicl inhiitor on either the mucosl or the serosl side (see elow). Osmotic grdients were eliminted y compenstion with mnnitol. The mmoni grdient, 1 mmol l 1 NH 4 Cl, used throughout the ssys is within the rnge experienced y this niml s plsm nd tissue vlues higher thn 15 mmol l 1 hve een mesured (Chew et l., 21; Moreir-Silv et l., 21; Tsui et l., 22). set of fluxes (N=3) ws lso mde without the ph-stt set-up nd se ws llowed to ccumulte in the mucosl sline over 2 h period. Totl HCO 3 nd CO 3 2 were then mesured s titrtle lklinity y doule end-point titrtion s descried previously (Grosell nd Genz, 26). In ddition, mmoni flux rtes were mesured following reversl of the mmoni grdient. fter the initil 1 h control flux, the 2 h experimentl flux with 1 mmol l 1 NH 4 Cl on the serosl side ws followed y finl 2 h with no mmoni on the serosl side nd 1 mmol l 1 NH 4 Cl on the mucosl side. Net se fluxes were not mesured in this series of experiments ecuse the serosl sline ws gssed with.3% CO 2 /O 2, which would hve interfered with the ph-stt titrtion. The inhiitors used were s follows (ll from Sigm-ldrich, St Louis, MO, US): ouin [1β,3β,5β,11α,14,19-hexhydroxycrd- 2(22)-enolide 3-(6-deoxy-α-L-mnnopyrnoside)]; DIDS (4,4 - diisothiocyno-2,2 -stilene-disulfonic cid); EIP [5-(N-ethyl-Nisopropyl) miloride]; nd ethoxzolmide (ETZ). Ouin is specific inhiitor of N + /K + -TPse (Silv et l., 1977), nd ws dded to the serosl sline t finl concentrtion of 1 mmol l 1 s 1 stock solution. DIDS is non-specific HCO 3 trnsport inhiitor tht hs een demonstrted to reduce Cl influx nd cuse lklosis y inhiiting Cl /HCO 3 exchnge (Cntchik et l., 1972), nd ws used in the mucosl sline t finl concentrtion of.1 nd 1. mmol l 1 s n 8 stock solution, crefully djusted to ph 7.8. EIP is potent inhiitor of NHE (Kleymn et l., 1988). In the present study, EIP ws dded to the mucosl sline t finl concentrtion of.1 mmol l 1 from 1 stock in vehicle [45% (w/v) 2-hydroxypropyl-β-cyclodextrin (.5% finl concentrtion)]. ETZ is perment C inhiitor nd ws used in the serosl sline t concentrtion of.1 mmol l 1 [2 (2 mmol l 1 ) stock solution in ethnol;.5% finl concentrtion] (Mren, 1977). ll the inhiitor stocks used in this study were lso recently used in similr in vitro studies on todfish nd trout, which confirmed their efficcy (Grosell nd Genz, 26; Grosell et l., 29; Genz et l., 211). During the course of the experiments, mucosl sline smples (2 μl) were tken every 3 min for mmoni mesurements nd replced with fresh mucosl sline. The serosl sline ws chnged t 2 h intervls, the only exception eing during the reversl experiment, when serosl sline smples were tken for the mesurement of mmoni flux in the reverse direction. Totl mmoni ws mesured s detiled elsewhere (Verdouw et l., 1978) nd mmoni flux rtes were clculted tking into considertion the effects of smpling on sline volume in the hlf chmers. The potentil interference of phrmcologicl gents nd vehicle in the ssy ws determined nd corrective mesures tken when pproprite. In initil tests, the commonly used vehicle DMSO ws found to interfere with the mmoni ssy nd ws, therefore, not used. mmoni flux rtes (J mm ) were clculted s: J mm = TN / ( t), (3) where TN represents the ccumultion of totl mmoni nitrogen in the mucosl sline (with the exception of the reverse grdient experiment, in which mmoni ws mesured in the serosl sline) corrected for time (t) in hours nd the two-dimensionl re () of the tissue insert (cm 2 ). mmoni flux rtes re expressed s μmol cm 2 h 1 nd positive vlues indicte net mmoni excretion into the mucosl sline. Sline [NH 3 ], [NH 4 + ] nd NH 3 prtil pressure (P NH3 ) were clculted from [TN ] nd ph mesurements using the Henderson Hssellch eqution with dissocition constnt (pk ) nd mmoni soluility coefficient (αnh 3 ) vlues for trout plsm t 25 C from Cmeron nd Heisler (Cmeron nd Heisler, 1983). The P NH3 grdient cross the intestinl preprtions ( P NH3 ) ws clculted s: P NH3 = P ser,nh3 P muc,nh3, (4) where P ser,nh3 is the P NH3 in the serosl sline nd P muc,nh3 is the P NH3 in the mucosl sline. Positive vlues fvor serosl to mucosl mmoni flux rtes. The Nernst potentil for NH 4 + (E NH4+ ) ws clculted s: E NH4+ = RTzF ln ([NH 4 + ] muc / [NH 4 + ] ser ), (5) where R is the gs constnt, T is the solute temperture, z is the vlency, F is Frdy s constnt, nd [NH 4 + ] ser nd [NH 4 + ] muc re the concentrtions of NH 4 + in the serosl sline nd mucosl sline, respectively. The true electrochemicl potentil or net driving force (F NH4+ ) for NH 4 + cross the intestinl epithelium ws clculted s: F NH4+ = E NH4+ TEP. (6) Positive F NH4+ vlues fvor serosl to mucosl mmoni flux nd negtive F NH4+ vlues fvor mucosl to serosl mmoni flux. Histologicl nlysis To ssess tissue integrity t the end of ech experiment, insert mounted tissues were fixed in situ in 3% prformldehyde/ phosphte-uffered sline (ph 7.3) overnight t 4 C. Tissue ws processed for prffin emedding, cross-sectioned (5 μm), nd stined with the periodic cid Schiff (PS) method, lcin lue (ph 2.5) nd 1% Gill s hemtoxylin (Merck, Whitehouse Sttion, NJ, US). Sections were viewed nd photogrphed with Leic DM6 photomicroscope. mmoni nd ir exposure experiment Fish (5.7±1.89 g nd 9.4±1.6 cm, mens ± s.d.) for tissue collection were divided into three groups (22 nimls in ech group) nd cclimted under control conditions (dechlorinted tp wter), high mmoni (3 mmol l 1 NH 4 Cl t ph 7.2 nd 21 C in dechlorinted tp wter, corresponding to 2 mmol l 1 NH 3 ) or eril exposure (1 ml of dechlorinted tp wter) for 7 dys, similr to conditions descried previously (Tsui et l., 22). t the end of the experiment the fish were overdosed with neutrlized tricne methnesulfonte [1:5 (w/v)] nd killed y decpittion; nterior nd posterior intestinl tissues were excised nd immeditely frozen in liquid nitrogen, nd stored t 8 C for posterior intestine memrne fluidity mesurements. Gut memrne fluidity Plsm memrnes from intestine were purified y density grdient centrifugtion (Dveloose et l., 1993). In rief, smples were pooled into three groups (ech group pooled from 7 8 smples) nd homogenized in isoltion medium (3 mmol l 1 sucrose, 1 mmol l 1 Tris-HCl, 1 mmol l 1 DTT), 1:4 (w:v), with glss Dounce homogenizer (pestle ) on ice. The memrnes were seprted on sucrose step grdient t 1, reltive centrifugl force (RCF; eckmn Coulter Optim Mx with n MLS-5 swinging-ucket THE JOURNL OF EXPERIMENTL IOLOGY

4 626 The Journl of Experimentl iology 216 (4) rotor; eckmn Coulter, Fullerton, C, US). To determine memrne enrichment nd purity, totl protein ws determined y the icinchoninic cid method (Smith et l., 1985) nd N + /K + - TPse nd lctte dehydrogense ctivities mesured ccording to estlished protocols (Ktynski et l., 24). Memrne fluidity ws mesured using fluorimetric method (fluorescence nisotropy) (Ktynski et l., 24). In rief, fluorescence nisotropy of the proe 1,6-diphenyl-1,3,5-hextrienyl-propionic cid (DPH) incuted with the memrnes ws mesured with POLRstr Glxy microplte fluorometer (MG Ltech, Frnkfurt, Germny), with excittion nd emission monochromtors set t 36 nd 43 nm, respectively, in temperture grdient (27, 29, 34, 37 nd 39 C). The nisotropy of the proe DPH gives n indiction of lipid order, with higher nisotropy corresponding to more ordered memrne. more ordered memrne is predicted to e less permele (Kikeri et l., 1989; Lnde et l., 1995; Ktynski et l., 24). Sttisticl nlysis Results re presented s mens ± s.e.m. Two-wy repeted mesures NOV followed y Student Newmn Keuls post hoc test ws used for electrophysiology, ph-stt nd mmoni flux nlysis ecuse two independent fctors (tissue nd tretment) ffected the tested response. For two group comprisons, t-tests were performed (SigmStt 3., SPSS, Chicgo, IL, US). Memrne fluidity liner regressions were compred y NCOV (R softwre). Vlues were considered significntly different t P<.5. RESULTS Electrophysiologicl properties of nterior nd posterior intestine The conductnce (G) nd short circuit current (I sc ) were significntly higher in the nterior intestine thn in the posterior intestine (2.5- nd 9.4-fold, respectively). No differences were found etween intestinl regions with regrds to the TEP (Tle 2). oth conductnce nd TEP were stle over the experimentl time course under current-clmp conditions in preliminry experiments (supplementry mteril Fig. S1). Only fter 5 h did TEP in the posterior intestine nd conductnce in the nterior intestine increse significntly reltive to the pre-tn control period. In greement, histologicl exmintion of the mounted tissue indicted tht tissue integrity ws mintined (Fig. 1). Net mmoni nd se flux In ll experiments, net mmoni flux rtes under control conditions were higher in nterior (.32±.7 μmol cm 2 h 1 ) thn in posterior intestine (.6±. μmol cm 2 h 1 ), nd in some cses flux rtes in the posterior intestine were elow the detection limit. In order to hve consistent mmoni efflux, 1 mmol l 1 TN ws dded to the serosl side, nd the net mmoni flux incresed significntly to 1.7±.3 μmol cm 2 h 1 in the nterior intestine, nd to.96±.26 μmol cm 2 h 1 in the posterior intestine. In the nterior Tle 2. Electrophysiologicl properties of the wetherloch nterior nd posterior intestine under symmetricl conditions during current clmp nd voltge clmp Intestinl region G (μsi cm 2 ) I sc (μ cm 2 ) TEP (mv) nterior 9.24± ± ±.29 Posterior 3.65± ± ±.83 P-vlue.8 < Vlues re mens ± s.e.m. (N=4) (t-test P-vlues re shown). G, conductnce; I sc, short-circuit current; TEP, trnsepithelil potentil. C Fig. 1. Histologicl sections of nterior (,,D) nd posterior (C,E) intestinl preprtions mounted in the Ussing chmers. Microgrphs C re from control preprtion nd microgrphs D E re from preprtions exposed to 1. mmol l 1 ouin. Sections re stined with lcin lue (ph 2.5), periodic cid Schiff nd Gils hemtoxylin. Scle rs, 1.5 mm (), 25 μm ( E). nd posterior intestine, respectively, the clculted serosl mucosl grdients for NH + 4 ( NH + 4 ) were 9.41±.7 nd 9.64±.6 mmol l 1, nd for NH 3 ( NH 3 ) they were.33±.2 nd.311±.1 mmol l 1. The mesured TEPs were.83±.63 nd.55±.68 mv, respectively, nd clculted equilirium + potentils for NH 4 (E NH4+ ) were 97.66±2.74 nd ±4.97 mv, respectively. The clculted NH + 4 driving forces (F NH4+ ) were 98.21±2.58 nd ±5.65 mv for nterior nd posterior intestine, respectively. These clcultions indicte strong serosl to mucosl grdients in oth preprtions. In preliminry experiments, the net mmoni flux remined stle over 4 h period (mximum length of experimentl protocols) t high serosl TN. ecuse of the reltively smll smple numer in individul experiments, ll the flux dt from control nd 1 mmol l 1 serosl mmoni periods re summrized in Tle 3. During the initil control flux, net se flux rtes were significntly greter in the nterior thn in the posterior intestine. ppliction of the 1 mmol l 1 TN serosl mucosl grdient resulted in significnt decrese in net se flux rte in the nterior intestine fter 2 h, while significnt increse ws oserved in the posterior intestine t oth 1 nd 2 h. There were no differences in se flux etween the nterior nd posterior intestine with the 1 mmol l 1 serosl mucosl mmoni grdient. In preliminry experiments, net se flux remined stle over 6 h period. Tle 3. Summry of se flux rtes (μequiv cm 2 h 1 ) under control nd serosl 1 mmol l 1 TN (1 nd 2 h) conditions pooled from inhiitor experiments D E TN Intestine region Control 1 h 2 h nterior.82± ±.49,.57±.58 Posterior.33±.125, *.567± ±.122 Dt were nlyzed y two-wy repeted mesures NOV (N=11). Within given tissue, groups with different uppercse/lowercse letters re significntly different. *Significnt difference from nterior intestine. TN, totl mmoni nitrogen. THE JOURNL OF EXPERIMENTL IOLOGY

5 Gut mmoni excretion nd lkliniztion mechnisms in loch 627 The HCO 3 flux rtes mesured y doule end-point titrtion of the ccumulted HCO 3 equivlents over 2 h period in the sence of ph-stt titrtion were.569±.11 nd.62±.129 μmol cm 2 h 1 for nterior nd posterior intestine, respectively. Rtes were significntly higher in the nterior intestine (P=.45; pired t-test). In the nterior intestine over 7% of overll se flux could e ccounted for y HCO 3 equivlents; however, in the posterior intestine less thn 1% ws ccounted for in this wy. fter 2 h, the mucosl ph incresed to 8.27±.31 nd 7.78±.91 in the nterior nd posterior intestine, respectively (P=.61) from n initil ph 7.742±.21. Phrmcologicl effects In the nterior intestine, the ddition of the N + /K + -TPse inhiitor ouin to the serosl sline, in the presence of the 1 mmol l 1 serosl mucosl TN grdient, cused significnt decrese in the net mmoni flux of 54% (Fig. 2). No significnt difference ws oserved in the posterior intestine. There were lso significnt decreses in the net se flux in the nterior intestine (37 46%) with no significnt chnges in the posterior intestine (Fig. 2). During this experiment TEP grdully decresed in oth intestinl regions (supplementry mteril Fig. S2). Ouin hd no effect on nterior intestine conductnce; however, in the posterior intestine, conductnce incresed significntly in the finl hour of tretment. The chloride trnsport inhiitor DIDS dded to the mucosl sline t dose of.1 mmol l 1 hd no effect on net mmoni flux in either the nterior or the posterior intestine (Fig. 3). However, the higher dose of 1. mmol l 1 DIDS inhiited the mmoni flux y 53% nd 87%, respectively. There ws significnt decrese in the net se flux in the nterior intestine of up to 66% during the course of DIDS exposure, while no effect ws oserved in the posterior intestine (Fig. 3). DIDS hd no effect on either TEP or conductnce in either intestinl region (supplementry mteril Fig. S3). The NHE-specific inhiitor EIP significntly decresed the mmoni flux rte in oth the nterior nd the posterior intestine y 74% nd 71%, respectively, when dded to the mucosl side (Fig. 4). No effect of EIP on se excretion ws detected in either intestinl region (Fig. 4). EIP hd no effect on conductnce in the nterior intestine ut in the posterior intestine, conductnce incresed grdully during the tretment. In the posterior intestine, TEP ws vrile during the course of the experiment ut the only significnt difference ws etween the 1 h EIP tretment nd 1 h TN groups. No significnt chnges in nterior intestine TEP were oserved (supplementry mteril Fig. S4). The C inhiitor ETZ hd no effect on net se flux (Fig. 5) in either the nterior or the posterior intestine. J mm dt re not presented, s loss of some smples prevented sttisticl nlysis. ETZ hd no effect on nterior intestine conductnce or TEP; however, in the posterior intestine, conductnce incresed mrkedly (supplementry mteril Fig. S5). No chnges in posterior intestine TEP were oserved. Reversl of the mmoni grdient Following the removl of the 1 mmol l 1 TN from the serosl side nd the ddition of mmoni with the sme concentrtion to the mucosl side, the NH 3 nd NH 4 + grdients nd F NH4 + were reversed (Tle 4). However, the solute mgnitude of the reversed NH 3 ws lower ecuse ph differences developed in the sence of the ph-stt system ut were still comprle etween intestinl regions. When compring the solute mgnitude of the net mmoni flux rtes, thus irrespective of direction (serosl mucosl versus mucosl serosl), there ws no difference in the nterior Net mmoni flux (µmol cm 2 h 1 ) mmol l 1 TN 1. mmol l 1 Ou 2. Net se flux (µequiv cm 2 h 1 ) nterior intestine Posterior intestine Control TN TN+Ou intestine. However, in the posterior intestine significnt decrese (49%) ws oserved (Fig. 6) even though NH 3 nd NH 4 + grdients nd F NH4 + would predict otherwise. mmoni grdient reversl resulted in opposite effects on epithelil conductnce with significnt decrese in the nterior intestine nd significnt increse in the posterior intestine (supplementry mteril Fig. S6). Posterior intestine TEP lso incresed significntly while no significnt difference ws oserved in the nterior intestine. Gut memrne fluidity nisotropy ws temperture dependent nd regressions of control, mmoni nd eril exposure were prllel. Posterior intestine fluorescence nisotropy ws found to e higher, thus indicting lower memrne fluidity, in the HE (intercept= , P<.2) nd ir-exposed fish (intercept= , P<.4) compred with control (intercept=.234). The insufficient numer of pooled nterior intestine smples did not llow us to determine sttisticl differences etween the exposure groups. However, trend similr to tht of the posterior intestine, with the regression lines of HE nd ir-exposed groups positioned ove the control regression line, indictes lower memrne fluidity thn controls s well. DISCUSSION mmoni trnsport involving the N + /K + -TPse nd NHE is demonstrted y the phrmcologicl inhiition of the net mmoni Pre-TN 1 h TN 2 h TN 1 h Ou 2 h Ou C D Fig. 2. Effect of serosl dditionl of the N + /K + -TPse inhiitor ouin (Ou, 1 mmol l 1 ) on () net mmoni flux rtes nd () net se flux rtes in the presence of 1 mmol l 1 totl mmoni nitrogen (TN) serosl mucosl grdient in Misgurnus nguillicudtus nterior nd posterior intestine. Vlues re mens + s.e.m. (N=4). rs with different uppercse/lowercse letters re significntly different (P<.5). D THE JOURNL OF EXPERIMENTL IOLOGY

6 628 The Journl of Experimentl iology 216 (4) Net mmoni flux (µmol cm 2 h 1 ) nterior intestine Posterior intestine Control TN TN+DIDS 1 mmol l 1 TN DIDS.1 mmol l 1 1. mmol l 1 Net se flux (µequiv cm 2 h 1 ) Pre-TN 1 h TN 2 h TN.1 mmol l 1 C C DIDS D 1. mmol l 1 DIDS Fig. 3. Effect of mucosl ddition of the chloride trnsport inhiitor DIDS (.1 nd 1. mmol l 1 ) on () net mmoni flux rte nd () net se flux rte in the presence of 1 mmol l 1 TN serosl mucosl grdient in M. nguillicudtus nterior nd posterior intestine. Vlues re mens + s.e.m. (N=4). rs with different uppercse/lowercse letters re significntly different (P<.5). Net mmoni flux (µmol cm 2 h 1 ) mmol l 1 TN.1 mmol l 1 EIP Net se flux (µequiv cm 2 h 1 ) Control TN TN+EIP Pre-TN 1 h TN 2 h TN 1 h EIP 2 h EIP nterior intestine Posterior intestine Fig. 4. Effect of mucosl ddition of the N + /H + exchnger inhiitor EIP (.1 mmol l 1 ) on () net mmoni flux rte nd () net se flux rte in the presence of 1 mmol l 1 TN serosl mucosl grdient in M. nguillicudtus nterior nd posterior intestine. Vlues re mens + s.e.m. (N=4). rs with different uppercse/lowercse letters re significntly different (P<.5). excretion rte with ouin nd EIP, respectively in the loch gut. The intestinl lumen lkliniztion cn e ttriuted to DIDS ( non-specific HCO 3 trnsport inhiitor)-sensitive net se (HCO 3 ) flux in the nterior intestine, n importnt component of the proposed mmoni voltiliztion mechnism. se flux under serosl mucosl mmoni grdient ws not dependent on C, s indicted y lck of inhiition with ETZ. Memrne fluidity of the posterior intestine ws lower in HE nd ir-exposed fish thn in controls, which correltes with lower mmoni memrne permeility, s ws evident from the lower mmoni flux rte in the reversed mmoni grdient experiment (see Fig. 7). In the present study, TEP in the nterior nd posterior intestine under symmetricl conditions ws not different; however, conductnce (G) nd I sc were higher in the former. These differences re in line with the striking morphologicl nd functionl differences in these regions, with the nterior intestine eing more ctive zone of the gut with cler digestive nd sorptive role nd the posterior intestine hving cler function in respirtory gs exchnge (McMhon nd urggren, 1987; Gonçlves et l., 27). These differences re lso in greement with the proposed trnsporterfcilitted NH 4 + excretion in the nterior intestine. The NHE is implicted in gut mmoni excretion s EIP (NHE inhiitor) decresed mmoni flux rte in oth the nterior nd the posterior intestine. lthough it ws not possile to mesure intestinl lumen N + levels in the loch, vlues mesured in low slinity (2.5 p.p.t.)-cclimted todfish Opsnus et (McDonld nd Grosell, 26) nd post-prndil freshwter tilpi (Grosell, 27) nd rinow trout (ucking nd Wood, 26; ucking nd Wood, 29) were within the rnge of the mucosl sline N + concentrtions used in this study. Thus, fvorle picl N + electrochemicl grdient would e present to drive the exchnge. While our study is the first to demonstrte role for fish intestinl NHE in mmoni excretion, there is mple evidence suggesting direct or indirect role of NHE in gill mmoni excretion in qutic orgnisms. In vivo exposure of Periophthlmodon schlosseri to 1 4 mol l 1 miloride ( non-specific NHE inhiitor) induced decrese in net mmoni excretion, indicting NHE involvement in mmoni excretion (Rndll et l., 1999). In Oncorhynchus mykiss exposed to.5 mmol l 1 nd 1. mmol l 1 miloride, decrese in mmoni excretion y 58% nd 87%, respectively, ws oserved ut not with lower dose of.1 mmol l 1 miloride (Lin nd Rndll, 1991). In study y Wilson nd co-workers, O. mykiss exposure to miloride (1 4 mmol l 1 ) did not indicte the presence of direct N + /NH 4 + exchnge; nevertheless, it decresed net mmoni flux y ~2% (Wilson et l., 1994). These studies were ll performed in vivo nd the inhiitors were dded to the wter, which implies tht they were ffecting gill picl NHE. However, similr in vivo inhiitor studies in loch with 1 4 mol l 1 miloride hd no effect on mmoni excretion rtes (Moreir-Silv et l., 21). Insted, s demonstrted in numer of other fishes, indirect coupling of n picl H + -TPse nd Rhesus (Rh) THE JOURNL OF EXPERIMENTL IOLOGY

7 Gut mmoni excretion nd lkliniztion mechnisms in loch 629 Net se flux (µequiv cm 2 h 1 ) nterior intestine Posterior intestine Pre-TN 1 h TN 2 h TN 1 h ETZ 2 h ETZ 1 mmol l 1 TN.1 mmol l 1 ETZ Fig. 5. Effect of serosl ddition of the cronic nhydrse inhiitor ETZ (.1 mmol l 1 ) on net se flux rte in the presence of 1 mmol l 1 TN serosl mucosl grdient in M. nguillicudtus nterior nd posterior intestine. Vlues re mens + s.e.m. (N=6). rs with different uppercse/lowercse letters re significntly different (P<.5). glycoprotein mmoni trnsporters fcilitte rnchil mmoni excretion (Weihruch et l., 29; Wright nd Wood, 29). However, recent studies in the yolk sc skin (surrogte gill ionocyte model) of lrvl fish give the est detiled mechnistic evidence of the role of the NHE in mmoni excretion (Shih et l., 28; Shih et l., 212; Wu et l., 21; Kumi nd Perry, 211). In zerfish lrve skin, lthough the H + -TPse still domintes the proton flux (8%) (Shih et l., 28), NHE3 gene knockdown nd EIP hve lso een shown to decrese the proton grdient tht drives fcilitted NH 3 diffusion y n cid-trpping mechnism through the picl Rh glycoprotein mmoni trnsporter Rhcg1 under low sodium (Shih et l., 212) nd low ph (Kumi nd Perry, 211) conditions when NHE3 expression is enhnced. Significntly, mmoni excretion hs een found to drive N + uptke vi NHE under these conditions (Shih et l., 212; Kumi nd Perry, 211). In contrst to zerfish, in lrvl medk skin, NHE is the dominte proton excretory mechnism linked to Rhcg1- medited mmoni excretion (Wu et l., 21). Evidence of direct N + nd NH 4 + exchnge vi NHE in fish gill is lcking lthough in mmmlin kidney proximl tuule, NHE3 functions directly in N + nd NH 4 + exchnge in the sence of RhCG expression (Weiner nd Verlnder, 211). In the loch intestine, it remins to e determined whether the EIP-sensitive NHE is cting directly in N + /NH 4 + exchnge or functions s N + /H + exchnger coupled to n picl Rh mmoni trnsporter. In the nterior intestine, ouin inhiition of N + /K + -TPse decresed net mmoni flux y 54%, demonstrting tht the ion trnsporter N + /K + -TPse is involved in mmoni excretion. This my e direct role through the solterl uptke of NH 4 + in plce of K + in exchnge for N + y the TPse, which hs een demonstrted in the gills of the gint mudskipper, P. schlosseri (Rndll et l., 1999), the oyster todfish, O. et (Mllery, 1983) nd the lue cr, Cllinectes orntus (Grçon et l., 27). However, N + /K + -TPse my ply n dditionl or lterntive indirect role through the mintennce of cell electronegtivity nd low intrcellulr N +. In this wy, the inwrd N + electrochemicl grdient would drive picl NH 4 + excretion vi NHEs. n increse in intestinl epithelil resistnce with ouin tretment in goldfish intestine hs een demonstrted nd linked to the collpse of the lterl intercellulr spces (lus et l., 1979), which might suggest similr effect in loch, wherey the prcellulr pthwy is locked therey decresing mmoni flux. However, in loch neither chnges in nterior intestine conductnce nor chnges in epithelil morphology (collpse of lterl intercellulr spces) were oserved with ouin tretment. lso, the increse in conductnce (decrese in resistnce) of the posterior intestine with ouin did not correlte with chnges in J mm. The preferentil effects of ouin in the nterior intestine re reflected y the oservtion tht tissue expression levels of N + /K + -TPse α-suunit, determined y immunolotting nd immunohistochemistry, re much higher in this gut region thn in the posterior intestine (Gonçlves et l., 27) (J.C.M.-S. nd J.M.W., unpulished oservtion). The se flux in the loch nterior nd posterior intestine in the sence of imposed mmoni grdients ws.82 nd.33 μequiv cm 2 h 1. lthough on the high side, these rtes re comprle to those mesured in mrine [.5 μequiv cm 2 h 1 (see Grosell, 21)] nd freshwter [.3 μequiv cm 2 h 1 (Genz et l., 211)] fishes. The high nterior intestinl flux rtes my reflect the fct tht the gut of the loch is stright nd very short, nd s the nterior region represents only one-fifth of this length, the trnsport cpcity must e ccommodted into reltively smll re. The findings were lso surprising given tht the loch is gstric nd, therefore, does not need intestinl se secretion to neutrlize gstric cid secretion s in the mrine species studied (Tylor et l., 211; Wilson et l., 211). DIDS ws effective in inhiiting HCO 3 excretion t 1 3 mol l 1 s hs een demonstrted in Pltichthys flesus (Grosell nd Jensen, 1999) nd O. mykiss (Grosell et l., 29), lthough in nguill jponic (ndo nd Surmnym, 199) nd Cithrichthys sordidus (Grosell et l., 21) the lower dose of 1 4 mol l 1 ws effective, ut not in Gillichthys mirilis (Dixon et l., 1986). t Tle 4. Clcultion of trnsepithelil grdients ΔNH + 4 *, ** ΔNH 3 * E NH 4 + ** TEP *, ** F NH 4 + ** (mmol l 1 ) (mmol l 1 ) (mv) (mv) nterior s m 9.1±.4.3± ± Posterior s m 9.27±.5.31± ± nterior m s ± ± ±17.5 Posterior m s ± ± ±1.86 Trnsepithelil grdients of NH 4 + (ΔNH 4 + ) nd NH 3 (ΔNH 3 ), nd the Nerst equilirium potentil (E NH 4 + ), trnsepithelil potentil (TEP) nd driving force (F NH 4 + ) cross the nterior nd posterior intestine preprtions with nominl 1 mmol l 1 TN serosl to mucosl (s m) nd reversed 1 mmol l 1 TN mucosl to serosl (m s) grdients. positive vlue for ΔNH 4 + or ΔNH 3 would fvor mmoni flux in the serosl mucosl direction, while negtive vlue would fvor mucosl serosl movement. Negtive F NH 4 + vlues would fvor serosl mucosl NH 4 + flux. Grdients were verged over the flux periods (N=4). solute dt vlues were nlyzed y 2-wy NOV. *Significnt difference etween serosl mucosl nd mucosl serosl grdients; **significnt difference etween nterior nd posterior intestine. THE JOURNL OF EXPERIMENTL IOLOGY

8 63 The Journl of Experimentl iology 216 (4) Net mmoni flux (µmol cm 2 h 1 ) Pre-TN Serosl mucosl Mucosl serosl the lower dose of 1 5 mol l 1 DIDS, inhiition of HCO 3 excretion ws lso not oserved in O. mykiss (Wilson et l., 1996). It hs een noted tht the preprtion of DIDS is key to its efficcy (Grosell et l., 29), nd such precutions were tken in the present study. We thus conclude tht the HCO 3 excretion mechnism is reltively DIDS insensitive in loch nd similr to tht seen in trout nd flounder (Grosell et l., 1999; Grosell et l., 29). The DIDS inhiition of the in vitro mmoni flux my in prt e explined y the indirect cid se effects on epithelil cells nd the picl oundry lyer. There is evidence tht DIDS cn inhiit mmoni flux indirectly through inhiition of the sodium icronte co-trnsporter (NC) in kidney medullry thick scending lim nd lung lveolr cells (Tokud et l., 27; nterior intestine Posterior intestine C, * Fig. 6. Effect of mmoni grdient reversl from 1 mmol l 1 TN serosl mucosl to 1 mmol l 1 TN mucosl serosl grdient on corresponding serosl mucosl nd mucosl serosl mmoni net flux rtes in M. nguillicudtus nterior nd posterior intestine. Note tht flux rtes re presented s solute vlues, nd re thus irrespective of direction. Dt re presented s mens + s.e.m. (N=3). rs with different uppercse/lowercse letters re significntly different (P<.5). *Significnt difference from nterior intestine. Lee et l., 21). In erythrocytes, Grci-Romeu nd collegues demonstrted DIDS-sensitive K + flux (Grci-Romeu et l., 1991) nd, given the promiscuity of K + trnsporters for NH 4 + (Knepper et l., 1989), DIDS my e cting directly on mmoni flux. lthough DIDS hs een shown to inhiit N + /K + -TPse ctivity in vitro (Felli et l., 1984), this would e unlikely in the present study ecuse of the inccessiility of cytosolic inding sites for DIDS, which is memrne impermele. In the wetherloch voltiliztion model, we predicted the involvement of cytosolic nd n extrcellulr C in the respective intrcellulr hydrtion of CO 2 to form HCO 3 nd the luminl dehydrtion of HCO 3 into CO 2. However, the lck of effect of ETZ, perment inhiitor tht would inhiit oth intrcellulr nd extrcellulr luminl C, rgues ginst role for these proteins in net se nd mmoni flux in the wetherloch, nd tht unctlyzed rtes of rection re sufficient under high mmoni conditions. In contrst, in the mrine todfish (Grosell nd Genz, 26) nd sewter-cclimted rinow trout (Grosell et l., 29), using ETZ Grosell nd collegues demonstrted tht cytosolic C nd, thus, ctlyzed endogenous HCO 3 production re importnt to HCO 3 secretion. ndo nd Surmnym reported similr findings in. jponic using cetzolmide, nother C inhiitor (ndo nd Surmnym, 199). However, in mrine teleosts HCO 3 secretion is significnt driver of Cl uptke nd thus osmoregultion, which is not the cse for teleosts living in fresh wter (Grosell, 21). s corollry, in study on the euryhline rinow trout, slinity cclimtion resulted in n increse in cytosolic nd memrne-ound C isoforms (Grosell et l., 27), which re involved in HCO 3 secretion (Grosell et l., 29). Nevertheless, C hs een loclized y enzyme histochemistry in the wetherloch digestive trct to oth the stomch (nterior intestine) nd posterior intestine (Chng et l., 26). Our results in the loch would indicte role for trnsepithelil HCO 3 secretion, mking use of plsm HCO 3 nd solterl trnsporter such s NC (Tokud et l., 27; Kurit et l., 28), which is indirectly supported y the finding of ouinsensitive (N + /K + -TPse inhiited) net se flux in the nterior intestine. Similr effects of ouin on intestinl se secretion hve een reported for the gulf todfish intestine (Grosell nd Genz, 26). In ddition, the unctlyzed rtes of CO 2 hydrtion my simply provide sufficient intrcellulr HCO 3 tht is not rte limiting Intestinl lumen oundry lyer Fig. 7. Proposed model for gut lkliniztion nd mmoni voltiliztion through the gut of M. nguillicudtus. The solterl N + /K + -TPse, nd picl N + /H + exchnger (NHE) nd Cl /HCO 3 nion exchnger re shown. predicted solterl N + :HCO 3 co-trnsporter is lso included. The moleculr identity of these trnsporters nd the potentil involvement of Rhesus proteins wit future work. Epithelium lood side THE JOURNL OF EXPERIMENTL IOLOGY

9 Gut mmoni excretion nd lkliniztion mechnisms in loch 631 to se flux. The possiility tht the high serosl mmoni levels resulted in n lkliniztion of the epithelium (NH 3 diffusion) would lso hve decresed the importnce of C y directly driving CO 2 hydrtion (NH 3 + CO 2 NH HCO 3 ). HE (2 mmol l 1 NH 3 ) exposure nd emersion oth resulted in lower memrne fluidity in the posterior intestine. This increse in memrne order would decrese pssive mmoni permeility, consistent with the results from the reversl of the 1 mmol l 1 TN grdient from serosl mucosl to mucosl serosl, which significntly reduced the mgnitude of the J mm. These findings gree with the prediction tht the intestinl epithelium hs low permeility to NH 3 in order to minimize pssive ck flux. Low mmoni permeility of numer of epitheli, including gstric glnds, kidney thick scending lim nd urinry ldder, hs een correlted with high memrne order (Lnde et l., 1995; Ktynski et l., 24; Kikeri et l., 1989; Singh et l., 1995). In other nimls, environmentl mmoni lso induced chnges in mmoni permeility t different sites such s the gill nd skin. Ip nd collegues, using different pproches (including in vitro mmoni flux rtes cross skin preprtion in n Ussing chmer system, nd cholesterol nd ftty cid content nlysis of skin), demonstrted tht P. schlosseri skin hs low permeility to NH 3 (Ip et l., 24). The low skin permeility to NH 3 is reflected in the ility of this fish to mintin low plsm levels of mmoni ginst lrge inwrd grdients in conjunction with ctive excretion of mmoni through the gills vi N + /K + (NH 4 + )-TPse nd NHE (Rndll et l., 1999). In summry, using n in vitro phrmcologicl pproch, we hve demonstrted tht the mechnism of mmoni excretion in the nterior intestine of the loch involves n picl EIP-sensitive NHE nd solterl N + /K + -TPse. The nterior intestine lso hs significnt net se secretion, which is lrgely HCO 3 - nd DIDSsensitive, indicting Cl /HCO 3 exchnge mechnism. These in vitro oservtions re consistent with the proposed mechnism of mmoni voltiliztion sed on in vivo mesurements (Tsui et l., 22). NH 4 + excreted y NHE-dependent mechnism into the lkline nterior intestinl oundry lyer forms gseous NH 3 nd H +. H + rects excreted HCO 3 forming gseous CO 2 nd oth these gses cn voltilize into the ir, pssing through the gut during intestinl rething nd thus voiding ph decrese ssocited with NH 3 formtion. The HCO 3 excretion lso mintins the lkline conditions (>ph 8) of the lumen surfce lyer, promoting NH 3 formtion. The moleculr identity of these trnsporters nd the potentil involvement of Rh proteins wit future work. LIST OF SYMOLS ND REVITIONS tissue re C cronic nhydrse DIDS 4,4 -diisothiocyno-2,2 -stilene-disulfonic cid DPH 1,6-diphenyl-1,3,5-hextrienyl-propionic cid EIP 5-(N-ethyl-N-isopropyl) miloride E NH Nernst potentil for NH 4 ETZ ethoxzolmide F Frdy s constnt F NH net driving force for NH 4 G conductnce HE high environmentl mmoni I current I sc short-circuit current J mm mmoni flux rte J se se secretion rte NC sodium icronte co-trnsporter NH 3 un-ionized mmoni + NH 4 mmonium ion NHE N + /H + exchnger pk P NH 3 R Rh t T TN TEP V z αnh 3 dissocition constnt un-ionized mmoni prtil pressure gs constnt Rhesus time solute temperture totl mmoni nitrogen trnsepithelil potentil electricl potentil vlency un-ionized mmoni soluility coefficient CKNOWLEDGEMENTS We would like Dr T. K. N. Tsui for procuring the fish nd H. Sntos nd R. Gerdes for fish mintennce support. J.M.S. is currently working t 3ʼs Reserch Group iomterils, iodegrdles nd iomimetics, University of Minho, Hedqurters of the Europen Institute of Excellence on Tissue Engineering nd Regenertive Medicine, ve Prk, Clds ds Tips, Guimrães, Portugl, I Institute for iotechnology nd ioengineering, PT Government ssocited Lortory, Guimrães, Portugl. FUNDING This work ws supported y Fundção pr Ciênci e Tecnologi (FCT) grnt [POCTI/SE/47585/22] to J.M.W. J.M.S. ws supported y n FCT scholrship [SFRH/D/1676/24] nd M.G. is supported y the Ntionl Science Foundtion (NSF) [grnt no. IOS ]. This reserch ws lso prtilly supported y the Europen Regionl Development Fund (ERDF) through the COMPETE Opertionl Competitiveness Progrmme nd ntionl funds through the Foundtion for Science nd Technology (FCT) under the project PEst-C/MR/L15/211. REFERENCES lus, H., Groot, J.. nd Siegeneek vn Heukelom, J. (1979). 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