RESEARCH ARTICLE Independence of net water flux from paracellular permeability in the intestine of Fundulus heteroclitus, a euryhaline teleost

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1 8 The Journl of Experimentl Biology 21, Pulished y The Compny of Biologists Ltd doi:1.1242/je.64 RESEARCH ARTICLE Independene of net wter flux from prellulr permeility in the intestine of Fundulus heterolitus, euryhline teleost Chris M. Wood 1,2, nd Mrtin Grosell 1 1 Rosenstiel Shool of Mrine nd Atmospheri Sienes, University of Mimi, Mimi, FL 33149, USA nd 2 Deprtment of Biology, MMster University, 128 Min Street West, Hmilton, ON, Cnd, L8S 4K1 Author for orrespondene (woodm@mmster.) Aepted 2 Novemer 211 SUMMARY Prellulr permeility nd sorptive wter flux ross the intestine of the euryhline killifish were investigted using in vitro gut s preprtions from sewter- nd freshwter-limted nimls. The permeility of polyethylene glyol (PEG), wellestlished prellulr proe, ws mesured using tre mounts of rdiolelled oligomers of three different moleulr sizes (PEG-4, PEG-9 nd PEG-4) t vrious times fter stition feeding. All three PEG moleules were sored, with permeility delining s liner funtion of inresing hydrodynmi rdius. Response ptterns were similr in sewter nd freshwter preprtions, though wter sorption nd PEG-9 permeility were greter in the ltter. Despite up to 4-fold vritions in sorptive wter flux ssoited with feeding nd fsting (highest t 1 3 h, lowest t h nd intermedite t 1 2 weeks post-feeding), there were no hnges in PEG permeility for ny size oligomer. When PEG permeility ws mesured in the opposite diretion (i.e. serosl to muosl) from net wter flux, it ws gin unhnged. HgCl 2 (1 3 mol l 1 ), puttive loker of quporins, eliminted sorptive wter flux yet inresed PEG-4 permeility y 6- to 8-fold in oth freshwter nd sewter preprtions. Experimentlly rising the serosl osmollity y ddition of 3 mmol l 1 mnnitol inresed the sorptive wter flux rte 1-fold, ut did not lter PEG permeility. Under these onditions, HgCl 2 redued sorptive wter flux y 6% nd gin inresed PEG permeility y 6- to 8-fold in oth freshwter nd sewter preprtions. Clerly, there ws no influene of solvent drg on PEG movement. The puttive prellulr loker 2,4,6-triminopyrimidine (TAP, 2 mmol l 1 ) hd no effet on net wter flux or PEG permeility. We onlude tht PEG nd wter move y seprte pthwys; sorptive wter trnsport proly ours vi trnsellulr route in the intestine of Fundulus heterolitus. Key words: prellulr permeility, trnsellulr permeility, feeding, fsting, Cl uptke, solvent drg, polyethylene glyol, PEG. INTRODUCTION As in most mrine teleosts (Grosell, 26), the intestine of the ommon killifish Fundulus heterolitus sors fluid tht is strongly hypertoni on net sis (Wood et l., 21). In reent yers, gret strides hve een mde in understnding the ion trnsport mehnisms tht help drive this wter sorption (reviewed y Wilson et l., 22; Wilson nd Grosell, 23; Grosell, 26; Grosell nd Tylor, 27; Grosell et l., 29; Grosell, 211; Grosell, 211). However, there hs een little progress on the eqully importnt question of how the wter tully moves. Is the net flux y trnsellulr route, prellulr route, or omintion of the two? In this regrd, the teleost intestine is not unusul. In reent review, Hill (Hill, 28) noted: The trnsport of fluid is one of the fundmentl proesses in physiology Despite voluminous literture, there is no ler ide of how it ours. Our investigtion of this prolem ws stimulted y two oservtions. Firstly, in the ourse of study on the impt of feeding on ion nd id se trnsport in the killifish gut (Wood et l., 21), we found tht there were lrge inreses in ion nd wter sorption immeditely fter feeding, whih hd ttenuted y h. Seondly, nd somewht surprisingly, these hnges tended to e lrger in the intestines of freshwter-limted killifish. We therefore exploited these nturl differenes in fluid sorption rtes, ssoited with feeding nd slinity, to see whether there were orresponding differenes in prellulr permeility, using three polyethylene glyol (PEG) oligomers of inresing size (PEG-4, PEG-9 nd PEG-4). PEG is well estlished s prellulr mrker in intestinl tissues (Pppenheimer nd Reiss, 1987; Wtson et l., 21). We resoned tht if some of the inresed sorptive wter flux ws prellulr, we would expet to find greter PEG permeility, espeilly if there ws onvetive solvent drg omponent to wter movement (Whittemury et l., 198; Pppenheimer nd Reiss, 1987; M et l., 1991). A differentil response with the different size oligomers of PEG might provide informtion on the size of the prtiulr prellulr hnnels involved (He et l., 1998; Wtson et l., 21). We followed up these experiments y mesuring PEG permeility in the serosl to muosl diretion, i.e. in the opposite diretion from net wter flux. We lso exmined PEG permeility under onditions of osmoti lmping, where the net muosl to serosl wter flux ws inresed 1-fold. The influene of HgCl 2, generl inhiitor of quporins in the trnsellulr pthwy (Cerd nd Finn, 21), ws exmined on wter flux nd PEG permeility under isosmoti onditions, nd under osmoti lmping. Finlly, we mesured PEG permeility nd wter flux in the presene of puttive loker of the prellulr pthwy, 2,4,6-triminopyrimidine (TAP) (Rehmn et l., 23). Our results tell simple story. There ppers to e solutely no effet of ltered net wter flux on PEG permeility, regrdless of

2 PEG permeility nd wter flux in killifish intestine 9 the size of the PEG moleules. HgCl 2 tretment redues or elimintes sorptive wter flux yet gretly inreses PEG permeility, wheres TAP hs no effet. We onlude tht sorptive wter trnsport is very proly y trnsellulr route in the intestine of F. heterolitus. MATERIALS AND METHODS Experimentl nimls Experiments were performed on gut s preprtions from ~34 ommon killifish of the northern suspeies Fundulus heterolitus mrolepidotus (Wlum 1792) (3 1 g). The fish were olleted y Aquti Reserh Orgnisms (ARO) Ltd (Hmpton, NH, USA) y eh-seining of lol tidl flts, nd then shipped to the University of Mimi where they were held for t lest 2 months prior to experiments. Alimtion nd experimentl temperture ws C. Killifish were kept in lrge groups (3 nimls per l tnk) in either freshwter or sewter. For freshwter limtion, slinity ws hnged grdully over 48 h in severl of the tnks, nd then the nimls were held in flowing freshwter for t lest 2 dys prior to test. The omposition of Mimi sewter (37. p.p.t.) nd freshwter hve een reported previously (Wood nd Grosell, 28). Additionl fish from the sme soure were shipped to MMster University for the phrmologil experiments. At MMster, they were held in the sme wter qulity (syntheti sewter of the sme slinity, or syntheti freshwter dupliting Mimi freshwter) s t Mimi, ut in filtered tnks in whih the wter ws renewed weekly. In oth freshwter nd sewter, the nimls were fed Aqu Mx sinking pellets (% protein) with the following mesured ioni omposition ( mol g 1 ): N + 23, Cl 17, C , Mg nd K + 326, t dily rtion of ~1.%. The stndrd protool ws to keep the fish on the 1.% dily rtion prior to n experiment, then fst them for 48 h, followed y dministrtion of stition mel. Test dissetions showed tht the ingested rtion ws typilly 3 4% of ody mss. The experiments were pproved y institutionl niml re ommittees t the University of Mimi nd MMster University. Stndrd gut s experiments In preliminry trils, we found tht net wter trnsport nd PEG permeility were the sme when mesured over 1, 2 nd 3 h periods, ut not therefter; stndrd 2 h period ws used in most experiments. Fish were killed for gut s experiments t 1 3 h post-feeding, h post-feeding, nd fter 7 14 dys of fsting in oth freshwter nd sewter. At eh time nd t eh slinity, flux experiments were performed on 6 9 different preprtions for eh of three different moleulr msses (4, 9, 4 D) of rdiolelled PEG ([ 3 H]PEG-4, [ 3 H]PEG-9, [ 14 C]PEG-4; Amerin Rdiohemils, St Louis, MO, USA). A modified Cortlnd sline (Wolf, 1963) ws used for ll gut s preprtions (mmol l 1 ): NCl 144, KCl.1, CCl 2 2H 2 O 1.4, MgSO 4 7H 2 O 1.9, NHCO , N 2 HPO nd gluose.. The osmollity ws 33 mosmol kg 1. The sline ws gssed with.3% CO 2 nd 99.7% O 2 mixture (i.e. nominl P CO2 2.2 Torr, P O2 >7 Torr, where 1 Torr 133 P) nd the ph ws djusted to 8.12 with smll mounts of 1 mol l 1 HCl. In eh gut s preprtion, one of the rdiolelled PEG ompounds ws pled in the muosl sline t onentrtion of. 1. Ci ml 1, representing mximum totl PEG onentrtion of. 1. g ml 1. Methods generlly followed those developed erlier (Sott et l., 26). Fish were lightly nesthetized in MS-222 (.1 g l 1 ), killed y ephli onussion, nd the intestinl trt disseted out while the fish ws kept old on ie. The killifish is gstri, with the oesophgus entering diretly into the nterior intestine through pylorus-like sphinter. The entire trt from this point k to the nus ws removed nd formed into single gut s preprtion. This omprises the nterior, mid nd posterior setions orresponding to the first, seond nd third portions in the digrm of Bkin nd Bowie (Bkin nd Bowie, 1928); the distl 1% of the trt (orresponding to the point where the posterior intestine joins the retum) ws exluded euse it ws found to e very frgile. The preprtion ws flushed thoroughly with ie-old sline to ler out food nd hyme. The distl end of the intestine ws then tied off nd het-flred theter (PE 9 polyethylene tuing) seured into the other end with 2- silk suture. The s ws then filled with rdiolelled sline (typilly 2 6 l depending on fish size), tking re not to trp ir ules, smple (1 l) of the filling sline ws tken for nlysis of initil muosl rdiotivity ([PEG m ] i ), nd the theter ws then seled with pin. The preprtion ws lotted on tissue pper in stndrdized mnner, weighed to.1 mg ury on n nlytil lne, then suspended in Flon TM tue ontining 11 ml of the sme sline (ut lking the rdiolelled PEG). The externl sline ws ontinully uled with the humidified gs mixture (99.7% O 2,.3% CO 2 ) nd onstnt temperture of 23 C ws mintined y wter th. After 1 min, ml smple ws tken for reording of initil serosl rdiotivity ([PEG s ] i ). A 2 h inution period ensued, during whih time the externl sline volume ws 6 ml. A finl ml smple of externl sline ws tken for reording of finl serosl rdiotivity ([PEG s ] f ). The preprtion ws lotted nd weighed extly s efore for grvimetri determintion of fluid trnsport rte. The ontents of the s were olleted for nlysis of finl muosl sline rdiotivity ([PEG s ] m ). The empty s ws then lotted dry nd weighed. Sutrtion of this mss from the initil nd finl filled msses yielded the initil (V i ) nd finl (V f ) muosl sline volumes, respetively. Finlly, the s ws ut open, wetted, strethed to mximum size on grph pper, nd the gross re (A) of the exposed epithelil surfe determined y tring its outline onto the pper (Grosell nd Jensen, 1999). All smples for mesurement of [ 3 H]PEG or [ 14 C]PEG rdiotivity were ounted in totl volume of ml sline plus 1 ml of Eolume fluor (MP Biomedils, St Louis, MO, USA). Tests showed tht quenh ws onstnt. Cl ws mesured y oulometri titrtion (Rdiometer-Copenhgen CMT 1, Copenhgen, Denmrk). Net wter trnsport rtes ( l m 2 h 1 ) were lulted from the differene etween initil (V i ) nd finl (V f ) muosl sline volumes, ftored y the intestinl surfe re (A, m 2 ) nd time (T, h). PEG permeility ws lulted s: ([PEG s ] f [PEG s ] i ) 6 P PEG =.([PEG m ] i + [PEG m ] f ) T 36 A, (1) where P PEG is in m s 1, PEG rdiotivities re in.p.m. ml 1, T is in h, A is in m 2, 6 refers to the externl volume in ml nd 36 onverts hours to seonds. The reltive PEG flux/wter flux rtio ws lulted s: ([PEG s ] f [PEG s ] i ) 6 Flux rtio =.([PEG m ] i + [PEG m ] f ) (V i V f ). (2) If the flux rtio is 1., PEG is moving t the sme reltive rte s wter. If it is greter thn 1., PEG is moving fster thn wter, nd if it is less thn 1., PEG is moving t slower rte thn wter. Reverse permeility gut s experiments In the stndrd gut s experiments, PEG permeility ws mesured y trking the flux of PEG rdiotivity in the sme diretion s the sorptive wter flux, i.e. from muosl to serosl omprtments. In ontrst, in this experiment, PEG permeility ws mesured y

3 1 C. M. Wood nd M. Grosell PEG permeility nd wter flux in killifish intestine trking PEG rdiotivity flux from serosl to muosl omprtments, i.e. in the opposite diretion from net wter flux. Experiments were performed only with 3 [H]PEG-4 nd 14 [C]PEG- 4 (N 6 8 per tretment) on preprtions tken t h postfeeding from sewter killifish. The preprtions were mde in the stndrd fshion, ut the rdiotive PEG ws dded t the norml onentrtion (. 1. Ci ml 1 ) to the serosl sline, whih hd volume of only 2 ml (otherwise vst mount of rdiotivity would hve een used). Simultneous ontrols were run where PEG permeility ws mesured in the stndrd fshion. Net wter flux ws lulted in the stndrd mnner, ut PEG permeility ws lulted from the pperne of rdiotivity in the muosl sline: P PEG = ([PEG m ] f V f ) ([PEG m ] i V i ).([PEG s ] i + [PEG s ] f T 36 A. (3) ) This pproh ws used in preferene to everted gut s preprtions, s initil trils indited tht this unnturl proedure resulted in dmge, mnifesting s gretly inresed PEG-4 permeilities nd deresed net wter trnsport rtes. Osmoti lmping gut s experiments Preprtions were mde in the stndrd fshion, ut 3 mmol l 1 of mnnitol ws dded to the serosl sline to elerte the fluid trnsport rte. This rised the mesured serosl osmollity from 33 to 8 mosmol kg 1. Simultneous ontrols were run where the serosl osmollity ws mintined t 33 mosmol kg 1. Experiments were performed only with 3 [H]PEG-4 nd 14 [C]PEG-4 (N 6 8 per tretment) on preprtions mde t h post-feeding from sewter killifish, whih exhiited the lowest endogenous wter sorption rtes t this time. As sorptive wter flux rtes were elevted in the experimentl tretment, the inution period ws shortened to 1 h. Net wter flux rtes nd PEG permeilities (Eqn 1) were lulted s outlined ove. HgCl 2 gut s experiments Meruri hloride (HgCl 2 ; Sigm-Aldrih, St Louis, MO, USA) is n effetive loker of most fish quporins (Cerd nd Finn, 21), inluding severl reently loned from F. heterolitus (Tingud- Sequeir et l., 29). Tests were performed with HgCl 2 onentrtions of either 1 4 or 1 3 mol l 1 on preprtions mde t h post-feeding from oth sewter killifish nd freshwter killifish (i.e. four seprte trils, N 7 11 per tretment). Gut s preprtions were mde in the stndrd mnner, ut were preinuted with HgCl 2 on the muosl surfe only for. h efore the flux mesurement egn. HgCl 2 remined present in the muosl sline during the flux mesurement. Controls were run simultneously with the sme pre-inution protool ut without HgCl 2. Net wter flux rtes nd PEG permeilities were lulted s ove. Osmoti lmping plus HgCl 2 gut s experiments The osmoti lmping nd HgCl 2 tretments were omined, using muosl HgCl 2 onentrtion of 1 3 mol l 1 nd serosl osmollity of 8 mosmol kg 1, hieved y the ddition of 3 mmol l 1 mnnitol to the serosl sline. The. h pre-inution with HgCl 2 on the muosl surfe ourred efore the serosl osmollity ws rised to 8 mosmol kg 1. Experiments were performed on gut ss mde t h post-feeding from oth sewter killifish nd freshwter killifish (i.e. two seprte trils, N 6 8 per tretment). Simultneous ontrols involved preinution nd exposure to high serosl osmollity, ut without the ddition of muosl HgCl 2. Net wter flux rtes nd PEG permeilities were lulted s ove. TAP gut s experiments The ompound TAP (Sigm-Aldrih) is puttive prellulr pthwy loker tht hs een reported to gretly redue wter sorption in the smll intestine of the rt (Rehmn et l., 23). TAP ws tested in four seprte series (N 6 9 per tretment) for its effets on wter sorption nd 14 [C]PEG-4 permeility in gut s preprtions from sewter killifish t 1 3 or h postfeeding. TAP ws dded to the muosl sline t onentrtion of 2 mmol l 1, whih is effetive in rt intestine (Rehmn et l., 23), either with or without 1 h pre-inution efore the flux mesurement ws strted. TAP remined present in the muosl sline during the flux mesurement. Mnnitol, t onentrtion of 2 mmol l 1, ws dded to the serosl sline to lne osmoti pressure. Controls with the sme onentrtions of mnnitol on oth surfes were run simultneously. Net wter flux rtes nd PEG permeilities were lulted s ove. Sttistil nlyses Dt re reported s mens ± s.e.m. (N numer of gut s preprtions). In few ses, dt were log-trnsformed so s to pss tests of normlity (Shpiro Wilk) nd homogeneity (Brtlett s hi squre) prior to further sttistil nlysis. One-wy ANOVA followed y Fisher s LSD test ws used for omprison of mens within slinity. Differenes etween freshwter nd sewter nimls t speifi times or etween speifi tretments were determined using Student s unpired t-test. Tests were two-tiled nd vlues were onsidered signifintly different t P<.. RESULTS Gut PEG permeility nd wter sorption in sewter killifish with respet to time post-feeding Permeility to PEG in preprtions from sewter fish ws essentilly independent of time post-feeding, ut strongly dependent on the moleulr size of the PEG oligomer (Fig. 1A). Permeility fell from out 1 18 ( 1 7 ) m s 1 for PEG-4 to 9 14 ( 1 7 ) m s 1 for PEG-9 nd to 1 3 ( 1 7 ) m s 1 for PEG-4. The only signifint effet of time post-feeding ws higher permeility to PEG-4 in fish tht hd een fsted for 1 2 weeks. This pttern ws ompletely different from the pttern in sorptive wter flux (Fig. 1B), whih ws gretest immeditely fter feeding (8 11 l m 2 h 1 ), t minimum t h post-feeding (2 3 l m 2 h 1 ), nd intermedite in long-term fsted fish (4 l m 2 h 1 ). Note tht net wter flux ws independent of the size of the PEG moleules, whih is to e expeted s only tre mounts of rdiolelled PEG were used. The disonnet of PEG permeility from wter sorption ws emphsized y lultion of the reltive PEG flux to net wter flux rtio (Fig. 1C). The rtio exhiited wide divergene from 1., eing well elow 1. t ll times for PEG-4, ut ove 1. for PEG- 4 nd PEG-9 t h post-feeding, the time of lowest net wter flux (Fig. 1B). Gut PEG permeility nd wter sorption in freshwter killifish with respet to time post-feeding Overll ptterns were very similr to those in sewter killifish, with PEG permeility gin eing independent of time post-feeding ut strongly dependent on the size of the PEG moleule (Fig. 2A). PEG permeility tended to e slightly higher in freshwter preprtions, ut the only signifint differene etween freshwter nd sewter fish ws higher permeility to PEG-4 in freshwter preprtions t 1 3 h post-feeding.

4 PEG permeility nd wter flux in killifish intestine 11 PEG permeility ( 1 7 m s 1 ) Wter flux (µl m 2 h 1 ) A B 4,,, d d ,,, Fig. 1. (A) Permeility of intestinl s preprtions from sewterlimted killifish to PEG-4, PEG-9 nd PEG-4 t 3 h, h nd 1 2 weeks fter stition mel. Permeility ws mesured in the muosl to serosl diretion. (B) Simultneously mesured sorptive flux of wter in the muosl to serosl diretion in these sme preprtions. (C) The reltive PEG flux to net wter flux rtio in these sme preprtions; if the flux rtio is >1., PEG is moving fster thn wter, nd if it is <1., PEG is moving t slower rte thn wter. See Mterils nd methods for detils. Mens + 1 s.e.m. (N 6 9 per tretment). Within pnel, mens shring the sme letter re not signifintly different from one nother (P>.). PEG flux/wter flux C ,, d Time fter feeding Agin, the pttern in net wter flux (Fig. 2B) ws ompletely different from the pttern in PEG permeility. Asorptive wter flux tended to e higher in freshwter preprtions, differenes tht were signifint in the PEG-4 tretment t 1 3 h post-feeding, nd in ll tretments t h post-feeding. Asorptive wter flux ws gin not ffeted y the size of the PEG moleules. Clultion of the reltive PEG flux to wter flux rtio (Fig. 2C) gin highlighted the lk of orreltion etween PEG permeility, whih ws reltively onstnt, nd net wter flux, whih vried gretly. Reverse permeility gut s experiments In this experiment with sewter preprtions t h postfeeding, PEG permeility ws mesured in the serosl to muosl diretion, opposite to the net sorptive flow of wter from muosl to serosl omprtments (Fig. 3A). Both PEG-4 permeility nd PEG-4 permeility were unhnged, regrdless of whether the PEG flux ws in the sme diretion or the opposite diretion to the net wter flux. Osmoti lmping gut s experiments When the osmollity of the serosl medium ws inresed from 33 to 8 mosmol kg 1 in sewter preprtions t h post-feeding, the rte of sorptive wter flux ws elerted y out 1-fold (Fig. 4A). This hd no effet on the permeility to PEG-4 or PEG-4 mesured in the sme diretion (Fig. 4B), gin highlighting the lk of oupling of PEG nd net wter flux. HgCl 2 gut s experiments The preeding results pointed to route for sorptive wter flux tht ws different from the prellulr PEG pthwy. Aquporins ould offer trnsellulr route, so the effet of the quporin loker HgCl 2, pplied muoslly, ws evluted. As quporin expression is reported to differ etween freshwter nd sewter teleosts (see Disussion), experiments were performed with preprtions from oth slinities t h post-feeding. At 1 4 mol l 1, HgCl 2 hd no effet on sorptive wter flux or PEG- 4 permeility in either freshwter or sewter gut ss (dt not shown). However, t 1 3 mol l 1, HgCl 2 effetively olished wter sorption (Fig. A) while inresing PEG-4 permeility y out 6-fold (Fig. B) in oth freshwter nd sewter preprtions, gin inditing omplete dissoition etween PEG nd wter flux. While these results might suggest n importnt role for quporins, it should e noted tht 1 3 mol l 1 HgCl 2 virtully eliminted sorptive Cl flux (freshwter: 297±72 nmol m 2 h 1 N 8 versus 1728±269 nmol m 2 h 1 N 11; sewter: 2±243 nmol m 2 h 1 N 7 versus 1194±72 nmol m 2 h 1 N 7). Thus, mjor driving fore for wter sorption ws lso inhiited. Osmoti lmping plus HgCl 2 gut s experiments The gol here ws to evlute the influene of HgCl 2 in sitution where n imposed osmoti grdient, rther thn Cl trnsport, would e the mjor driving fore for wter sorption.

5 12 C. M. Wood nd M. Grosell PEG permeility nd wter flux in killifish intestine PEG permeility ( 1 7 m s 1 ) Wter flux (µl m 2 h 1 ) PEG flux/wter flux A B C,,, ,,,,, Time fter feeding Fig. 2. (A) Permeility of intestinl s preprtions from freshwter-limted killifish to PEG-4, PEG-9 nd PEG-4 t 3 h, h nd 1 2 weeks fter stition mel. Permeility ws mesured in the muosl to serosl diretion. (B) Simultneously mesured sorptive flux of wter in the muosl to serosl diretion in these sme preprtions. (C) The reltive PEG flux to net wter flux rtio in these sme preprtions; if the flux rtio is >1., PEG is moving fster thn wter, nd if it is <1., PEG is moving t slower rte thn wter. See Mterils nd methods for detils. Mens + 1 s.e.m. (N 6 9 per tretment). Asterisks indite signifint differenes (P<.) from the omprle tretments in sewter preprtions of Fig. 1. Within pnel, mens shring the sme letter re not signifintly different from one nother (P>.). In ontrol experiments, the elevted serosl osmoti pressure of 8 mosmol kg 1 resulted in high, lmost identil sorptive wter flux in freshwter nd sewter gut ss (Fig. 6A), similr to those mesured in the erlier series with sewter preprtions (Fig. 4A). These very similr, very high wter flux rtes under osmoti lmping (Fig. 6A) ourred despite the greter net wter flux rtes in ontrol freshwter preprtions thn in ontrol sewter preprtions under isosmoti onditions (Fig. 1B, Fig. 2B, Fig. A). The effets of 1 4 mol l 1 HgCl 2 in omintion with high serosl osmollity were lso identil t the two slinities, resulting in 6% redution of wter flux (Fig. 6A) nd 6- to 8-fold elevtion in PEG-4 permeility (Fig. 6B) in oth ses. Therefore, one gin, wter nd PEG flux hnged in opposite diretions. HgCl 2 tretment lso redued, ut did not eliminte, sorptive Cl flux in oth freshwter (1823±77 nmol m 2 h 1 N 8 versus 424±438 nmol m 2 h 1 N 8) nd sewter preprtions (1928±138 nmol m 2 h 1 N 8 versus 419±24 nmol m 2 h 1 N 8) under osmoti lmping. TAP gut s experiments All four experiments with this puttive prellulr pthwy loker in the muosl omprtment yielded the sme result, so one only is reported in Tle 1 Regrdless of whether the 2 mmol l 1 TAP ws dded utely or for 1 h of pre-inution, or tested t 1 3 or h post-feeding, there were no signifint effets of the drug on net wter flux rtes, net Cl sorption rtes or PEG-4 permeility. DISCUSSION Overview The present results lerly dissoite the net movement of wter from the movement of the prellulr permeility mrkers PEG- 4, PEG-9 nd PEG-4 in the intestine of the killifish. Lrge hnges in sorptive wter flux hieved endogenously vi feeding nd fsting (Fig. 1B, Fig. 2B) or exogenously vi osmoti lmping (Fig. 4) hd essentilly no effet on PEG permeility, regrdless of moleulr size. Even when PEG permeility ws mesured y its flux ounter-urrent to tht of wter, the vlues remined unhnged (Fig. 3). A tretment (1 3 mol l 1 HgCl 2 ) tht gretly inresed PEG permeility, tully eliminted (Fig. ) or gretly redued (Fig. 6) sorptive wter flux. Clerly, there is no orreltion etween PEG movement nd net wter movement, nd no solvent drg effet. We onlude tht sorptive wter trnsport must our y pthwy entirely different from tht of PEG, nd tht is very likely to e vi trnsellulr route involving quporins. Although it seems unlikely, we nnot eliminte the possiility tht the generl

6 PEG permeility nd wter flux in killifish intestine 13 Wter flux (μl m 2 h 1 ) A PEG-4 PEG-4 Wter flux (μl m 2 h 1 ) A PEG-4 PEG-4 PEG permeility ( 1 7 m s 1 ) B M S M S M S M S S M M S Fig. 3. The influene of the diretion of mesurement on PEG permeility in intestinl s preprtions from sewter-limted killifish t h fter stition mel. (A) Asorptive wter flux, mesured in the muosl to serosl (MrS) diretion, ompnying the respetive PEG permeility mesurements in B. (B) Simultneous PEG-4 permeility (left pnel) nd PEG-4 permeility (right pnel) mesured y trking PEG rdiotivity flux in MrS or SrM diretions, i.e. in the sme diretion or in the opposite diretion reltive to the net sorptive flow of wter from muosl to serosl omprtments (MrS). Mens + 1 s.e.m. (N 6 8 per tretment). There were no signifint differenes with respet to the diretion of mesurement. lk of hnge in PEG permeility in ll tretments exept HgCl 2 my reflet the ft tht there is prellulr wter pthwy entirely seprte from the prellulr PEG pthwy, hene the finding of no solvent drg. Diffusive unidiretionl fluxes in dynmi equilirium through the prellulr pthwy lso remin possile, ut would not impt on the physiology of the fish. PEG permeility did tend to e higher in freshwter preprtions, nd freshwter preprtions tended to hve higher net wter flux (Figs 2 nd ). However, overll this differene in PEG permeility ws signifint for only one size of PEG moleule (PEG-9), s disussed elow (see Fig. 7). Very proly, this reflets different funtionl properties of the gut in freshwter versus sewter killifish with respet to Cl uptke, id se sttus nd the need for divlent ion rrier funtions in sewter fish, rther thn inditing funtionl reltionship etween net wter flux nd PEG permeility. It is noteworthy tht under osmoti lmping (ontrol tretments of Fig. 6), the pity for elevted net wter sorption ws the sme in freshwter nd sewter preprtions. This supports the view tht under isosmoti onditions, the higher net wter sorption in freshwter gut ss reflets funtionl regultion, rther thn differene in pity. One other differene ws higher permeility to PEG-4 in sewter fish tht hd een fsted for 1 2 weeks (Fig. 1A). In future studies, it would e of interest to exmine whether long-term fsting is ssoited with inresed lekiness to other moleules. M S S M PEG permeility ( 1 7 m s 1 ) B Control High OP Control High OP Control High OP Control High OP Fig. 4. The influene of rising the serosl osmollity from 33 mosmol kg 1 (Control) to 8 mosmol kg 1 (high osmoti pressure, High OP), y the ddition of 3 mmol l 1 mnnitol, on (A) the sorptive flux of wter nd (B) the simultneously mesured permeility to PEG-4 nd PEG-4 in the muosl to serosl diretion in intestinl s preprtions from sewter-limted killifish t h fter stition mel. Mens + 1 s.e.m. (N 6 8 per tretment). Asterisks indite signifint differenes from Control (P<.). TAP hd no effet on either PEG permeility or sorptive wter flux (Tle 1); this oservtion neither supports nor opposes our onlusions, ut this lk of effet is y no mens unpreedented. For exmple, in rit nd frog gll ldder preprtions, where there ws strong evidene for prellulr permeility to severl smll moleules, TAP did not ffet net wter or Cl flux rtes, or the permeility to surose, moleule of omprle size to PEG- 4 (Moreno, 197). The uthor onluded tht wter ws permeting minly vi the trnsellulr route. This is very different from mmmlin intestine, where TAP strongly redues permeility to wter nd numerous smll moleules, suggesting lrge prellulr flow of oth (Rehmn et l., 23). PEG permeility s funtion of moleulr size The hydrodynmi rdii (r, in Å) of PEG moleules re known (Ruddy nd Hdzij, 1992) nd n e lulted from moleulr msses (M) y the reltionship: r.29m.44. (4) Thus the men hydrodynmi rdii of PEG-4, PEG-9 nd PEG- 4 re 4.49, 6.36 nd 12.2 Å. As PEG permeility ws essentilly independent of time post-feeding (Fig. 1A, Fig. 2A), men permeility for ll tretments hs een plotted ginst men hydrodynmi rdius for freshwter nd sewter preprtions in Fig. 7. There ppers to e lose to liner negtive reltionship

7 14 C. M. Wood nd M. Grosell PEG permeility nd wter flux in killifish intestine Wter flux (µl m 2 h 1 ) PEG-4 permeility ( 1 7 m s 1 ) 12 A B Control HgCl 2 Control HgCl 2 Fig.. The influene of 1 3 mol l 1 HgCl 2, pplied only to the muosl surfe, on (A) the sorptive flux of wter nd (B) the simultneously mesured permeility to PEG-4 in the muosl to serosl diretion in intestinl s preprtions from freshwter-limted (left-hnd pnels) nd sewter-limted killifish (right-hnd pnels) t h fter stition mel. Mens + 1 s.e.m. (N 6 8 per tretment). Asterisks indite signifint differenes from Control (P<.). There were no signifint differenes etween freshwter nd sewter preprtions under the sme tretment onditions. Wter flux (µl m 2 h 1 ) PEG-4 permeility ( 1 7 m s 1 ) 4 A B 1 1 Control HgCl 2 Control HgCl 2 Fig. 6. The influene of 1 3 mol l 1 HgCl 2 pplied only to the muosl surfe, in omintion with high serosl osmollity (8 mosmol kg 1, hieved y the ddition of 3 mmol l 1 mnnitol), on (A) the sorptive flux of wter nd (B) the simultneously mesured permeility to PEG-4 in the muosl to serosl diretion in intestinl s preprtions from freshwter-limted (left-hnd pnels) nd sewter-limted killifish (right-hnd pnels) t h fter stition mel. The ontrols were sujeted only to high serosl osmollity. Mens + 1 s.e.m. (N 6 8 per tretment). Asterisks indite signifint differenes from Control (P<.). There were no signifint differenes etween freshwter nd sewter preprtions under the sme tretment onditions. etween PEG permeility nd hydrodynmi rdius, with the reltionship eing somewht elevted (i.e. higher permeility) in the freshwter fish. This differene ws signifint only t hydrodynmi rdius of 6.36 Å, i.e. for PEG-9, nd ws not seen in Fig. 2A euse of the lower N vlue in the individul tretments. These reltionships suggest severl onlusions. Firstly, y pplition of the Stokes Einstein eqution, simple free diffusion of the PEG moleules would e inversely proportionl to their hydrodynmi rdius. Thus, for free diffusion, PEG-4 permeility (4.49 Å) would e 2.79 times greter thn PEG-4 permeility (12.2 Å) nd PEG-9 permeility (6.36 Å) would e 1.98 times greter thn PEG-4 permeility. The tul differenes re fr greter thn this (8.- nd.7-fold, respetively, in sewter preprtions, nd 7.3-fold, respetively, in freshwter preprtions). Thus the prellulr rrier is signifint nd size seletive. Seondly, there ppers to e rnge of prellulr pore sizes in the intestine of the killifish, nd this rnge is not dissimilr to tht in the mmmlin smll intestine sed on omprle PEG permetion studies (M et l., 1991; He et l., 1998). Shep nd ollegues, using very different methods, rehed the sme onlusion out pore size in the intestine of the hinook slmon (Shep et l., 1997). Thirdly, there ppers to e greter undne of pores in the 6.36 Å rnge in freshwter killifish thn in sewter preprtions. Finlly, the tomi rdius of the wter moleule itself is only 1.3 Å, smller thn ll these pore sizes, so it remins possile tht there exists prellulr pthwy for wter tht would not intert with the prellulr pthwys for the vrious sized PEG moleules. For this lst sheme to explin the present Tle 1. The influene of 2 mmol l 1 2,4,6-triminopyrimidine (TAP) in the muosl solution on sorptive wter flux rtes, sorptive Cl flux rtes nd permeility to PEG-4 in intestinl s preprtions from sewter-limted killifish Control (N 6) TAP (N 6) Wter flux rte ( l m 2 h 1 ).32± ±1.33 Cl flux rte (nmol m 2 h 1 ) 1412± ±19 PEG-4 permeility ( 1 7 m s 1 ) 7.33± ±1.72 Dt re mens ± s.e.m. nd were otined h post-feeding. Permeility to PEG-4 ws mesured in the muosl to serosl diretion. There were no signifint differenes (P>.).

8 PEG permeility nd wter flux in killifish intestine 1 Permeility ( 1 7 m s 1 ) Freshwter Sewter Hydrodynmi rdius (Å) Fig. 7. Reltionships etween the mesured permeility to PEG nd the hydrodynmi rdius of the PEG moleules in intestinl s preprtions from sewter- nd freshwter-limted killifish. Men permeility for ll times post-feeding hve een plotted ginst men hydrodynmi rdius (PEG-4, 4.49 Å; PEG-9, 6.36 Å; nd PEG-4, 12.2 Å). Mens ± 1 s.e.m. (N 19 26). Asterisk indites signifint differene (P<.) etween freshwter nd sewter preprtions t the sme hydrodynmi rdius. dt, the lrger prellulr PEG pores would hve to e low in numer reltive to the prellulr wter hnnels nd would hve to seletively exlude wter moleules, so tht there ould e no oserved solvent drg effets. PEG-4 hs een extensively used s drinking rte mrker in fish y mny ls, inluding our own (e.g. Grosell et l., 1999), sine its introdution for this purpose (see Shehdeh nd Gordon, 1969), on the ssumption tht it is not sored ross the fish intestine (e.g. Skdhuge, 1974). However Boge nd ollegues (Boge et l., 1988) reported tht it ws lost t n ppreile rte from n intestinl perfuste in intt trout, nd the present study shows tht it is sored in vitro y the killifish intestine, leit t low rte. This should e tken into ount in future studies of drinking in fish nd in studies employing PEG s non-sorle mrker for wter trnsport in isolted intestinl segments. A role for quporins in intestinl wter sorption in fish The hnnels y whih trnsellulr wter sorption ours in the intestine of the eyuryhline killifish re proly quporins. Very reent work (reviewed y Cerd nd Finn, 21; Grosell, 211) shows tht mny different quporins re widely expressed in the intestinl trt of euryhline teleosts, nd tht AQP1 in prtiulr my ply role in wter sorption. Oservtions of elevted mrna expression following sewter trnsfer, elevted AQP1 protein undne in intestinl tissue following sewter limtion nd, finlly, pil s well s solterl loliztion of the protein suggest role in trnsepithelil wter movement (Aoki et l., 23; Mrtinez et l., 2; Mrtinez et l., 2; Rldu et l., 28). It hs een demonstrted tht AQP1 genes from fish onfer wter permeility in expression systems (MIver et l., 29), inluding AQP1 from F. heterolitus (Tingud-Sequeir et l., 29), ut despite the ove oservtions, diret evidene for role for AQP1 in intestinl wter sorption hs een lking. Consistent with this view, Cerd nd Finn ution tht the reltive ontriution of quporin-medited trnsellulr flux versus prellulr flux is lrgely unknown (Cerd nd Finn, 21). In the present experiments, HgCl 2 ws highly effetive inhiitor of wter sorption in killifish intestine (Figs nd 6), onsistent with the known ility of Hg 2+ to use ovlent modifition of ysteine within the seletivity filter region of the quporin hnnel pore, resulting in steri lokde (Svge nd Stroud, 27). The onentrtion needed to hieve full inhiition ws 1 3 mol l 1, the sme s tht required to inhiit fish quporins in n ooyte expression system (MIver et l., 29). However, 1 3 mol l 1 HgCl 2 hd two other notle effets: 6- to 8-fold elevtion of PEG- 4 permeility, nd n olition of sorptive Cl flux. The former provides further support for the onlusion tht wter flux is not ssoited with the prellulr pthwy, nd ords with studies on mmmlin intestine where HgCl 2 similrly inreses permeility to prellulr mrkers (Stirling, 197; Bohme et l., 1992; Kierel et l., 2). Presumly Hg 2+ interts with ysteine residues or other SH groups tht re ruil in limiting juntionl permeility. However, the omplete lokde of Cl sorption onfounds interprettion of the inhiition of net wter flux, euse the two mjor mehnisms thought to provide the osmoti linkge for wter flux under isosmoti onditions re oupled N + plus Cl o-trnsport, nd Cl /HCO 3 exhnge (Grosell, 26; Grosell, 211; Grosell, 211). The explntion is proly the known poteny of Hg 2+ in inhiiting N +,K + -ATPse nd other ATPses (Mush et l., 199; Lkshmi et l., 1991) tht power these mehnisms. Clerly, elimintion of net Cl trnsport provides n lterntive or dditionl explntion to lokde of quporins for the tion of HgCl 2 on sorptive wter flux. The tretment with HgCl 2 in omintion with high serosl osmollity (Fig. 6) ws designed to overome this onfounding issue y reting sitution in whih net wter flux would not e primrily driven y Cl trnsport. Agin HgCl 2 rised PEG-4 permeility y 6- to 8-fold, nd used similr inhiition of the now elevted sorptive wter flux (y ~6%) nd sorptive Cl flux (y ~%). However, s shown y the nlysis in Tle 2, with high serosl osmollity, the pprent Cl onentrtion in the sorte ws redued from its norml hypertoni level (~228 mmol l 1 ) (see lso Wood et l., 21) to vlues (~128 mmol l 1 ) elow tht Tle 2. The influene of high serosl osmollity lone or in the presene of muosl HgCl 2 on the onentrtion of Cl in the sorte of gut s preprtions from freshwter- or sewter-limted killifish Freshwter (mmol l 1 Cl) Sewter (mmol l 1 Cl) Inution sline Control ±13.2 x (1) 22.1±16.8 (13) High serosl osmollity 122.4±4.6 y (8) 133.2±4.3 (8) High serosl osmollity + HgCl ±4.7 z (8) 142.±. (8) Dt re mens ± s.e.m. (N) nd were otined h fter stition mel. The Cl onentrtion in the sorte ws estimted y dividing the mesured Cl sorption rte y the mesured net wter sorption rte in eh preprtion. The Cl onentrtion in the inution sline is shown for omprison. High serosl osmollity, 8 mosmol kg 1, hieved y the ddition of 3 mmol l 1 mnnitol; muosl HgCl 2 onentrtion, 1 3 mol l 1. For given slinity, mens shring the sme letter re not signifintly different (P>.). There were no signifint differenes (P<.) etween freshwter nd sewter vlues under the sme tretment onditions.

9 16 C. M. Wood nd M. Grosell PEG permeility nd wter flux in killifish intestine (12 mmol l 1 ) of the inution sline. Clerly, under these onditions, Cl sorption ws no longer driving wter sorption, yet the ltter ws still strongly inhiited y HgCl 2. Additionlly, it is noteworthy tht the pprent Cl onentrtion of the sorte rose fter HgCl 2 tretment (signifint in freshwter preprtions only, Tle 2), suggesting preferentil inhiition of net wter flux over net Cl flux. Kierel nd ollegues reported similr strong inhiition of sorptive wter flux in response to mol l 1 HgCl 2 in rt nd rit intestinl preprtions sujeted to elevted serosl osmollity (Kierel et l., 2). Differenes etween euryhline fish nd mmmls We re wre of no omprle nlysis of the routes of net wter flux in the intestine of teleost fish. However, there is preedent for ompletely trnsellulr flow of wter in n eletrilly leky epithelium. Using diret visuliztion tehnique, Kovsnjuk nd ollegues reported tht wter did not flow through the prellulr pthwy in n MDCK (renl) ell epithelium, even when osmoti grdients were pplied (Kovsnjuk et l., 1998). However, the sitution in the killifish gut ppers to e very different from tht in the mmmlin gut where muh of the inresed intestinl wter sorption ompnying mel ppers to e prellulr, nd serves to drive the prellulr sorption of ions nd nutrients y onvetive solvent drg, t miniml metoli ost (Pppenheimer nd Reiss, 1987; Mdr nd Pppenheimer, 1987). Indeed, reent review (Bkke et l., 211) noted tht the prellulr uptke of nutrients is generlly onsidered to e negligile in fish. In euryhline teleosts, the need for preise ontrol of ion nd wter sorption my e muh greter thn in mmmls euse of the intense nd vrile environmentl grdients. Here, intestinl sorption must serve ionond osmo-regultory requirements in ddition to the need for nutrient sorption. Therefore, this preision my e hieved y running the flux of oth wter nd ions/nutrients (vi speifi hnnels nd trnsporters) through the trnsellulr route, therey more esily ontrolling the mount of wter tht follows osmolyte uptke. One of the enefits of routing ll sorptive wter flux through the trnsellulr pthwy is tht it will void unwnted ion uptke y solvent drg. For exmple, Ndell nd ollegues demonstrted tht sorption of the essentil ut toxi ion Cu 2+ ws unrelted to solvent drg, osmoti pressure or hnges in trnsepithelil potentil in the gut of the euryhline rinow trout (Ndell et l., 27). Similrly, the uptke of other essentil ut toxi ions tht re t high levels in ingested sewter (Mg 2+, SO 4 2 ) (Grosell, 26; Grosell, 211) n e minimized y ensuring tht ll ion nd wter flux is routed through trnsellulr pthwys. Reent findings show tht hnges in slinity lter mrna expression of multiple isoforms for ludin 3, 1 nd 2 in the intestinl epithelium of euryhline fish (Clellnd et l., 21; Tipsmrk et l., 21), whih suggests tht these ludins my e involved in ontrolling nd regulting the prellulr pthwy nd thus potentilly the rrier funtions of the epithelium ginst movement of divlent ions. Of prtiulr interest is tht ertin isoforms re expressed more roustly in the distl segments where luminl serosl Mg 2+, SO 4 2 nd HCO 3 grdients re the steepest (Mrshll nd Grosell, 26). It is lso noteworthy tht quporin expression ppers to e gretest in the hindgut nd retum of severl speies (reviewed y Cerd nd Finn, 21). Our killifish intestine preprtions exluded prt of this re euse of its frgility (see Mterils nd methods), so it is possile tht our dt underestimte the wter sorption pity of the whole gut. It is lso possile, indeed likely, tht our dt sed on lmost whole intestinl ss hide regionl differenes in trnsport nd permeility long the trt. The need for preise ontrol of prellulr ion movement, espeilly in mrine fish, my e even more intense in the killifish euse of its unusul defiit in freshwter: in ontrst to N + uptke, there is no tive uptke mehnism for Cl t the gills (Ptrik et l., 1997; Ptrik nd Wood, 1999; Wood nd Lurent, 23), so this mjor nd essentil nion must e quired exlusively from the diet. Proly for this reson, intestinl Cl (s well s N + nd H 2 O) uptke rtes inrese during limtion to freshwter (Sott et l., 26), nd persist t higher rtes thn in sewter-limted nimls (Sott et l., 28; Wood et l., 21). The differene eomes more mrked t 1 3 nd h fter mel when Cl uptke rte, HCO 3 seretion rte (to whih Cl uptke is prtilly oupled) nd wter sorption re ll signifintly greter in freshwter killifish (Wood et l., 21). The sme differenes etween freshwter nd sewter preprtions t h fter the mel, t lest for Cl nd wter sorption, were seen in the present study. Notly, the Cl onentrtion of the sorte inreses with time of fsting, s would e predited if there is mehnism to ontrol how muh wter flux follows ion flux through the trnsellulr pthwy. Interestingly, the only other euryhline teleost tht is known to rely on intestinl rther thn rnhil Cl uptke in freshwter is the eel (Kirsh, 1972; Grosell et l., 2), nd here too the onentrtion of the sorte ws higher in freshwter thn in sewter nimls (Skdhuge, 1969). Additionl experiments (Skdhuge, 1969; Skdhuge, 1974) demonstrted tht the intestinl wter sorption in the eel ws losely linked to, nd presumly driven y, the tive trnsport of N + nd Cl, though the uthor suggested tht this might our vi the lterl interellulr spes (i.e. prellulr route), in ontrst to the urrent evidene. Beuse Cl uptke is oupled, t lest in prt, to HCO 3 seretion, nother onsequene of the higher Cl nd wter sorption rtes ross the intestine in freshwter killifish is higher ph in the gut lumen, oth in vitro nd in vivo (Wood et l., 21). Prellulr permeility is known to e ph sensitive (e.g. Powell et l., 1981), so the tendeny for higher PEG permeility in preprtions from freshwter nimls, signifint for PEG-9 (6.36 Å; Fig. 7), my reflet this id se differene. As n HClsereting stomh is entirely lking in F. heterolitus (Bkin nd Bowie, 1928), digestion must rely exlusively on mehnil nd enzymti hydrolysis. The produts my e different euse of the higher ph of the hyme, requiring different pore sizes for the prellulr sorption of lrge nutrients (e.g. polypeptides) in freshwter versus sewter killifish. In ddition, teleost quporins re known to e more permele to wter t higher ph vlues (reviewed y Cerd nd Finn, 21), so this my explin the higher wter sorption rtes in freshwter killifish (Fig. 2B). Clerly, there re mny questions tht n e pursued using this reltively simple experimentl system. ACKNOWLEDGEMENTS We thnk Lind Dio for exellent tehnil ssistne t MMster University, nd Dnielle MDonld for ess to equipment in Mimi. We lso thnk the nonymous reviewers for onstrutive omments, espeilly the suggestion of the HgCl 2 experiment. FUNDING This work ws supported y Nturl Sienes nd Engineering Reserh Counil of Cnd (NSERC) Disovery Grnt [to C.M.W.] nd Ntionl Siene Foundtion (NSF) grnt [IOB to M.G.]. C.M.W. is supported y the Cnd Reserh Chir Progrm.

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