Exposure to air, but not seawater, increases the glutamine content and the glutamine synthetase activity in the marsh clam Polymesoda expansa

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1 The Journl of Experimentl Biology 27, Pulished y The Compny of Biologists 24 doi:1.1242/je Exposure to ir, ut not sewter, increses the glutmine content nd the glutmine synthetse ctivity in the mrsh clm Polymesod expns Kum C. Hiong 1, Wendy Y. X. Peh 1, Ai M. Loong 1, Wi P. Wong 1, Shit F. Chew 2 nd Yuen K. Ip 1, * 1 Deprtment of Biologicl Sciences, Ntionl University of Singpore, 1 Kent Ridge Rod, Singpore , Repulic of Singpore nd 2 Nturl Sciences nd Science Eduction, Ntionl Institute of Eduction, Nnyng Technologicl University, 1 Nnyng Wlk, Singpore , Repulic of Singpore *Author for correspondence (e-mil: dsipyk@nus.edu.sg) Accepted 5 Octoer 24 Polymesod expns spends considerle portion of its life exposed to ir in mngrove swmps where slinity fluctutes gretly. Thus, the im of this study ws to evlute the effects of eril exposure (trnsfer from 1 rckish wter directly to ir) or slinity chnges (trnsfer from 1 rckish wter directly to 3 sewter) on nitrogen metolism in P. expns. We concluded tht P. expns is non-ureogenic ecuse crmoyl phosphte (CPS) III ctivity ws undetectle in the dductor muscle, foot muscle, heptopncres nd mntle when exposed to rckish wter (control), sewter or ir for 17 dys. It is mmonotelic s it excretes nitrogenous wstes minly s mmoni in rckish wter or sewter. After trnsfer to sewter for 17 dys, the contents of totl free mino cids (TFAA) in the dductor muscle, foot muscle, heptopncres nd mntle incresed significntly. This could e relted to n increse in protein degrdtion ecuse exposure to sewter led to greter rte of mmoni excretion on dys 15 nd 17, despite unchnged tissue mmoni contents. Alnine ws the mjor free mino cid (FAA) in P. expns. The contriution of lnine to the TFAA pool in vrious tissues incresed from 43 48% in rckish wter to 62 73% in sewter. In contrst, in clms exposed to ir for 17 dys Summry there were no chnges in lnine content in ny of the tissues studied. Thus, the functionl role of lnine in P. expns is minly connected with intrcellulr osmoregultion. Although % of the TFAA pool of P. expns ws ttriutle to glutmine, the glutmine contents in the dductor muscle, foot muscle, heptopncres nd mntle were unffected y 17 dys of exposure to sewter. However, fter exposure to ir for 17 dys, there were significnt increses in mmoni content in ll these tissues in P. expns, ccompnied y significnt increses in glutmine content (2.9-, 2.5-, 4.5- nd 3.4-fold, respectively). Simultneously, there were significnt increses in glutmine synthetse ctivities in the dductor muscle (1.56-fold) nd heptopncres (3.8- fold). This is the first report on the ccumultion of glutmine ssocited with n upregultion of glutmine synthetse in ivlve species in response to eril exposure, nd these results revel tht the evolution of glutmine synthesis s mens for detoxifiction of mmoni first occurred mong invertertes. Key words: evolution, lnine, glutmine, glutmine synthetse, clm, Polymesod expns. Introduction While ivlves re considered to e qutic nimls, mrsh clms Polymesod spp. spend considerle portion of their lives exposed to ir during low tides. For instnce, Polymesod crolinin is found most undntly in res of mrsh in north Florid tht re covered in wter for only 12% of the yer (Duoinis-Gry nd Hckney, 1982). In Singpore, relted species, Polymesod expns, is commonly found in chnnels nd pools formed y smll strems running etween the roots of mngrove plnts in the lndwrd fringe of mngroves. During spring tides the slinity of the pool is round 2 nd t nep tides etween 11 nd 2 (Morton, 1988). However, P. expns my not lwys e exposed to rckish wter, ecuse it is rrely covered y the tides in its nturl hitt. In the lortory, it cn withstnd considerle periods (up to months) of eril exposure. When exposed to ir, lower intertidl nd sutidl ivlves usully close their shells nd shift to neroic metolic pthwys (Widdows et l., 1979; de Zwn, 1983). In contrst, P. expns gpes nd slightly exposes the mntle mrgins posteriorly during eril exposure (Morton, 1988). Adduction of the vlves immeditely following emersion nd t periodic intervls helps to ventilte the mntle cvity (McMhon, 1988), nd gs exchnge occurs lrgely cross the surfce of the mntle. Such n dpttion sustins eroic metolism in

2 466 K. C. Hiong nd others the tissues of Polymesod spp. during long periods of eril exposure. Thus, P. crolinin is cple of the sme rte of O 2 uptke whether in ir or sumerged in sewter (Deton, 1991). The ir-rething cpcity of P. crolinin is exceptionl mong ivlve species found in high intertidl hitts; consequently, there is no mrked rdycrdi nd no evidence of ccumultion of metolites indictive of neroic metolism in this mrsh clm during emersion (Deton, 1991). Although P. expns is not confronted with hypoxi during long-term eril exposure, the moiliztion of proteins nd mino cids for energy supply would led to the relese of mmoni (Bishop et l., 1983). Indeed, mmoni ccumultes in the hemolymph nd mntle cvity fluid of Mytilus edulis exposed to ir (Shick et l., 1988). Since mmoni is toxic, terrestril ivlves like P. expns hve to defend themselves ginst mmoni toxicity during long-term emersion. Therefore, this study ws undertken to determine the effects of eril exposure (17 dys) on nitrogen metolism in P. expns. In order to confirm tht the effects nd phenomen oserved relted specificlly to eril exposure, we lso determined the effects of slinity chnges (from 1 to 3 ) on this mrsh clm for direct comprison. Ammoni is detoxified to ure s n excretory product in mny vertertes, including few fish species, the mjority of mphiins, some reptiles nd ll mmmls (Cmpell, 1973). The synthesis of excretory ure in certin lnd plnri (Cmpell, 1965), erthworms (Bishop nd Cmpell, 1963, 1965) nd snils (Cmpell nd Bishop, 197; Cmpell nd Speeg, 1968; Trmell nd Cmpell, 1972) vi the rginine ornithine ure cycle (OUC) indictes tht the modifiction of the sic nutritionl pthwy of rginine synthesis for mmoni detoxifiction first took plce mong inverterte nimls (Cmpell, 1973). However, Andrews nd Reid (1972) were unle to detect ctivities of crmoyl phosphte synthetse III (CPS III), crucil enzyme of the OUC, in the tissues of severl ivlve species (Mytilus cliforninus, Anodont kennerlgi, Sxidomus gignteus nd Compsomyx sudiphn). At present, no ivlve is known to possess functionl OUC (Bishop et l., 1983). Thus, the hypothesis tested in this study ws tht, like other ivlves, CPS III ws sent from the tissues of P. expns, nd tht this mrsh clm ws incple of detoxifying mmoni to ure during eril exposure despite the hrsh environment conditions in its nturl hitt. In mmmlin rins, mmoni toxicity cn e meliorted trnsiently through the ction of glutmine synthetse (GS), resulting in the synthesis of glutmine. The ccumultion of glutmine consequently leds to strocyte swelling nd rin dmge (Brusilow, 22). The cpcity for detoxifiction of mmoni to glutmine in higher vertertes cn e trced ck to fish. Severl species of tropicl ir-rething fishes cn detoxify mmoni to glutmine not only in the rin, ut lso in liver, muscle, stomch nd gut, during exposure to terrestril conditions or environmentl mmoni (Peng et l., 1998; Jow et l., 1999; Ip et l., 21, 24; Chew et l., 21; Anderson et l., 22; Ty et l., 23; Lim et l., 24). In contrst, glutmine is not known to e ccumulted s product of mmoni detoxifiction in tissues of invertertes, lthough it functions s minor osmolyte in certin species (Livingstone, 1985). Trcer studies using 14 C-glucose with clms, mussels, oysters, nd pulmonte nd prosornch snils indicte tht ll these molluscs hve strong cpcity for rpid iosynthesis of glutmte, lnine nd sprtte, ut the cpcity for glutmine iosynthesis is wek or nonexistent (see review y Bishop et l., 1983). Glutmine synthesis is energy dependent; one mole of ATP is hydrolysed for ech mole of mide-n formed. To dte, no ivlve is known to ccumulte glutmine during emersion, ecuse ivlves usully shift to neroic energy metolism nd ccumulte lnine, nd sometimes lnopine, during eril exposure (Bishop et l., 1983). P. expns is cple of withstnding eril exposure without undergoing neroiosis or reduction in metolic rte, so it is n idel specimen to study whether the cpcity for the detoxifiction of mmoni to glutmine first evolved mong invertertes. Therefore, in this study, we imed to verify tht glutmine ccumultion occurred in ssocition with incresed mmoni levels in clms exposed to terrestril conditions ut not in those exposed to sewter, when mmoni could e excreted freely. We lso imed to demonstrte tht glutmine ccumultion occurred in ssocition with n upregultion of GS ctivities in tissues of clms exposed to ir. Mterils nd methods Animls Specimens of Polymesod expns Mousson 1849 ( g) were collected from mudflts of the mngrove swmp t Krnji, Singpore. They were mintined in 15 l of rckish wter (1 ) with ertion in glss quri (43 cm 28 cm 3 cm, L W H) nd fed newly htched Artemi slin. Wter ws chnged every 3 dys. The clms were cclimtized to lortory conditions for t lest 1 week efore, nd food ws withdrwn 3 dys prior to, experimenttion. Determintion of wet mss of tissues The mss of clm (with shells) ws otined using Liror EB-28M lnce (Shimdzu, Kyoto, Jpn) to the nerest.1 g. It ws then forced open to dissect out the heptopncres, dductor muscle, foot muscle nd mntle. Results otined from individul tissues nd orgns were expressed s percentge of totl clm mss (with shells). To determine the wter content in tissue smples, the wet msses were recorded to the nerest.1 g. They were then dried in n oven t 95 C until constnt mss nd the dry mss recorded. The wter content of smple ws clculted s differences etween the wet mss nd the dry mss, nd expressed s percent of wet mss tissue.

3 Glutmine synthesis in P. expns 467 Experimentl conditions For exposure to sewter, groups of 1 clms were trnsferred from rckish wter directly to full strength sewter (3 ) in glss quri (43 cm 28 cm 3 cm, L W H). They were kept in sewter for 17 dys, during which no food ws supplied nd wter ws chnged dily. For eril exposure, groups of 1 clms were exposed to terrestril conditions in n uncovered dry glss qurium for 17 dys (87 9% humidity). Clms kept in rckish wter nd fsted for the sme period served s controls. On dy 17, clms were forced open nd the hemolymph ws collected from the foot muscle with syringe nd needle. After tht, the dductor muscle ws cut. The heptopncres, mntle, dductor muscle nd foot muscle were dissected nd freeze-clmped with liquid nitrogen-precooled luminium tongs. Hemolymph smples were deproteinized in two volumes of trichlorocetic cid (TCA) nd then centrifuged t 4 g for 15 min to otin the superntnt. Smples collected were kept t 8 C until nlysis. Determintion of mmoni nd ure excretion rtes To determine the rtes of mmoni nd ure excretion in P. expns in wter, clms were weighed nd kept individully in 12 ml of rckish wter (1 ) or sewter (3 ) with slight ertion in cylindricl continers (7.5 cm 8.5 cm, D H), nd the externl medi were chnged dily. On dys 3, 6, 9, 12 nd 17, wter smples (2 ml) were collected nd cidified with 2 µl of 2 mol l 1 HCl for mmoni nd ure nlyses. Smples were kept t 4 C nd nlyses were done within week. Ammoni ws determined ccording to the method of Anderson nd Little (1986). Ure content ws nlyzed s descried y Jow et l. (1999). Rtes of excretion re presented s µmol dy 1 g 1 clm (with shell). No ttempt ws mde to determine the rtes of mmoni nd ure excretion in clms exposed to ir. Although certin mounts of mmoni nd ure could e excreted into the mntle cvity fluid during eril exposure, the fluid ws kept within the niml nd ws in direct contct with vrious tissues. Enzyme ssys The heptopncres, mntle, dductor muscle nd foot muscle were homogenized three times in 5 volumes (w/v) of ice-cold extrction uffer contining 5 mmol l 1 Hepes (ph 7.6), 5 mmol l 1 KCl nd.5 mmol l 1 EDTA, using n Ultr Turrx (Jnke nd Kundel, Stufeni, Stufen, Germny) homogeniser t 24 revs min 1 for 2 s ech seprted y intervls of 1 s off. The homogente ws centrifuged t 1 g nd 4 C for 15 min to otin the superntnt, which ws susequently pssed through 1 ml Bio-Rd P-6DG column (Bio-Rd Lortories, Hercules, CA, USA) equilirted with ice-cold extrction uffer without EDTA. The filtrte ws used directly for enzyme ssy. CPS III (E.C ) ctivity ws determined in the presence of glutmine, N-cetylglutmte nd uridine triphosphte s descried y Anderson nd Wlsh (1995). Rdioctivity ws mesured using Wllc 1414 liquid scintilltion counter (Wllc, Oy, Finlnd). The CPS III ctivity ws expressed s µmol [ 14 C]- ure formed min 1 g 1 wet mss. GS (E.C ) trnsferse ctivity ws ssyed ccording to the method of Shnkr nd Anderson (1985). The formtion of γ-glutmylhydroxymte ws determined t 5 nm using Shimdzu UV 16 recording spectrophotometer. The GS ctivity ws expressed s µmol γ-glutmylhydroxymte formed min 1 g 1 wet mss. Determintion of mmoni, ure nd free mino cids (FAAs) The frozen smple ws weighed, ground to powder in liquid nitrogen, nd homogenized three times in 5 volumes (w/v) of 6% TCA using n Ultr-Turrx homogenizer t 24 revs min 1 for 2 s ech seprted y intervls of 1 s off. The homogente ws centrifuged t 1 g nd 4 C for 15 min to otin the superntnt. For mmoni determintion, the ph of the superntnt ws djusted to with 2 mol l 1 KHCO 3. Ammoni ws determined ccording to the methods of Bergmeyer nd Beutler (1985). Ure ws determined s descried y Jow et l. (1999). The difference in sornce of the smple with nd without urese tretment ws used to estimte the ure concentrtion in the smple. For FAA nlysis, the superntnt otined ws djusted to ph 2.2 with 4 mol l 1 lithium hydroxide nd diluted ppropritely with.2 mol l 1 lithium citrte uffer (ph 2.2). FAAs were nlyzed using Shimdzu LC-6A mino cid nlysis system (Kyoto, Jpn) with Shim-pck ISC-7/S154 Li-type column. Although complete free mino cid nlysis ws performed on every smple, only the contents of lnine, glutmte, glutmine, glycine, turine nd totl FAA (TFAA) re presented in this report. Results re expressed s µmol g 1 wet mss tissue or µmol ml 1 hemolymph, s pproprite. Sttisticl nlyses Results re presented s mens ± the stndrd error of the men (S.E.M.). Student s t-test or nlysis of vrince (ANOVA) followed y multiple comprisons using Duncn s procedure ws used to evlute differences etween mens in groups where pproprite. Arsine trnsformtion ws pplied to percentge results efore sttisticl nlyses. Differences where P<.5 were regrded s sttisticlly significnt. Results Exposure to sewter or ir for 17 dys hd no significnt effects on the wter contents of the dductor muscle, foot muscle, heptopncres nd mntle of P. expns (Tle 1). Ammoni ws excreted y P. expns during the 17 dys of exposure to rckish wter or sewter, ut no ure ws detected in the externl medi (Fig. 1). On dy 17, there ws significnt increse in the rte of mmoni excretion in clms exposed to rckish wter (Fig. 1). For clms exposed to sewter, there were significnt increses in rtes of mmoni excretion on dy 15 nd dy 17, nd these rtes were

4 468 K. C. Hiong nd others Tle 1. Effects of exposure to rckish wter (1 ; control) sewter (3 ) or ir (emersion) for 17 dys on the wter content in vrious tissues of Polymesod expns Wter content (% wet mss) Brckish wter Tissue (control) Sewter Emersion Adductor muscle 86.9± ± ±1.6 Foot muscle 83.3± ± ±.48 Heptopncres 87.7± ± ±1.91 Mntle 9.4± ± ± ,,, Vlues re mens ± S.E.M. (N=3)..6 1, Ammoni excretion rte (µmol dy 1 g 1 ) ,,c,d,e *,,,c,d *,,,c,d,e Dy Fig. 1. Time course (17 dys) of the effects of exposure to rckish wter (1, control; white rs) or sewter (3 ; grey rs) on the rte (µmol dy 1 g 1 clm) of mmoni excretion in Polymesod expns. Vlues re mens ± S.E.M. (N=5). *Significntly different from the corresponding rckish wter vlue (P<.5); significntly different from the dy-3 vlue (P<.5); significntly different from the dy-6 vlue (P<.5); c significntly different from the dy-9 vlue (P<.5); d significntly different from the dy-12 vlue (P<.5); e significntly different from the dy-15 vlue (P<.5). significntly greter thn those of the corresponding control in rckish wter (Fig. 1). There were no detectle CPS III ctivities (detection limit=.1 µmol min 1 g 1 tissue) in heptopncres, mntle, dductor muscle nd foot muscle of P. expns kept in rckish wter, sewter or terrestril conditions for 17 dys. In spite of high ckground sornce redings, tretments of smples with urese reveled tht no ure ws present in these tissues. Ammoni contents in the dductor muscle, foot muscle, heptopncres, mntle nd hemolymph of P. expns kept in sewter for 17 dys were comprle to those of clms kept in rckish wter (control) for the sme period (Fig. 2). In contrst, there were significntly greter levels of mmoni in ll these tissues in clms fter 17 dys of eril exposure Brckish wter Sewter Emersion Fig. 2. Effects of exposure to rckish wter (1, control), sewter (3 ) or ir (emersion) for 17 dys on mmoni contents in the dductor muscle (white rs), foot muscle (grey rs), heptopncres (lck rs), mntle (htched rs) nd plsm (dotted rs) of Polymesod expns. Vlues re mens ± S.E.M.(N=5). Significntly different from the rckish wter vlue (P<.5); significntly different from the sewter vlue (P<.5). compred with those exposed to rckish wter or sewter (Fig. 2). The contents of lnine in the dductor muscle, foot muscle, heptopncres nd mntle (Fig. 3) were greter thn those of glycine (Fig. 4), glutmte (Fig. 5) nd glutmine (Fig. 6) in P. expns exposed to ll three experimentl conditions. Turine content in the dductor muscle, foot muscle, heptopncres nd mntle of clms in rckish wter s 2.67±.31, 2.34±.55, 2.3±.48,.76±.5, respectively. Thus, lnine ws mjor contriutor to the TFAA pool (Fig. 7) in P. expns. The contents of lnine (Fig. 3) nd glycine (Fig. 4) in the dductor muscle, foot muscle, heptopncres nd mntle in clms exposed to sewter for 17 dys were significntly greter thn those in clms exposed to rckish wter or terrestril conditions for similr period. Exposure to sewter induced greter glutmte content only in the heptopncres nd mntle (Fig. 5), nd hd no effect on the glutmine content of ny of the tissues studied (Fig. 6). In contrst, eril exposure for 17 dys resulted in significntly greter glutmine content in the dductor muscle, foot muscle, heptopncres nd mntle of P. expns (Fig. 6). Both sewter nd ir exposure hd only minor effects on turine content in these tissues (results not shown). Overll, exposure to sewter, ut not to ir, led to significntly greter TFAA levels in ll the tissues of P. expns exmined s compred with exposure to rckish wter (Fig. 7). GS (trnsferse) ctivities were detected in the dductor muscle, foot muscle, heptopncres nd mntle of P. expns (Fig. 8). The ctivities of GS in these tissues in P. expns were unffected y exposure to sewter for 17 dys (Fig. 8),

5 Glutmine synthesis in P. expns Brckish wter Sewter Emersion Fig. 3. Effects of exposure to rckish wter (1, control), sewter (3 ) or ir (emersion) for 17 dys on the lnine contents in the dductor muscle (white rs), foot muscle (grey rs), heptopncres (lck rs) nd mntle (htched rs) of Polymesod expns. Vlues re mens ± S.E.M. (N=4). Significntly different from the rckish wter vlue (P<.5); significntly different from the sewter vlue (P<.5). Brckish wter Sewter Emersion Fig. 4. Effects of exposure to rckish wter (1, control), sewter (3 ) or ir (emersion) for 17 dys on the glycine contents in the dductor muscle (white rs), foot muscle (grey rs), heptopncres (lck rs) nd mntle (htched rs) of Polymesod expns. Vlues re mens ± S.E.M. (N=4). Significntly different from the rckish wter vlue (P<.5); significntly different from the sewter vlue (P<.5). ut eril exposure led to significnt greter ctivities of GS in the dductor muscle nd heptopncres (Fig. 8). Discussion P. expns in rckish wter There ws no detectle CPS III ctivity in the heptopncres, mntle, dductor muscle or foot muscle of P. expns. Thus, similr to other ivlves (Andrews nd Reid, 1972), P. expns is non-ureogenic. In ddition, no ure ws detected in the mient rckish wter. Therefore, P. expns is mmonotelic, excreting nitrogenous wstes minly s mmoni. FAAs re sustrtes for energy metolism, protein synthesis nd osmoregultion. Chnges in slinity nd/or exposure to environmentl contminnts my ffect the concentrtion nd composition of FAAs in ivlves (Byne et l., 1976,; Livingstone, 1985). It hs een reported tht the FAA levels in the tissues of rckish wter ivlves (e.g. P. crolinin nd Rngi cunet) contriute 25 3% of the intrcellulr solute t 1 2 slinity (Allen, 1961; Fyhn, 1976; Giney, 1978,; Henry et l., 198), nd lnine is the mjor contriutor to the FAA pool (Virkr nd We, 197; Pierce, 1971; Giney, 1978; Henry et l., 198; Mtsushim et l., 1984). Indeed, similr to other ivlves, lnine (43 48%) ws the mjor FAA in P. expns in rckish wter (lso in sewter nd in ir). More importntly, our results confirm tht glutmine contriuted significntly ( %) to the TFAA pool of P. expns in rckish wter. This contriution ws greter thn tht of glycine ( %) nd comprle to tht of glutmte ( %). The high levels of glutmine in the tissues of P. expns indicte tht this mrsh clm ws cple of synthesizing glutmine. Indeed, we verified the presence of GS (trnsferse) ctivities in ll the tissues exmined, with the gretest ctivity in the heptopncres. Due to the pucity of informtion in this re, generliztions out glutmine iosynthetic cpilities of ivlves cnnot e mde. However, studies with 14 C-lelled precursor molecules (glutmte, Kres cycle intermedites nd glucose) in M. edulis nd other ivlve species confirm the sence of ny glutmine iosynthetic cpcity (Wijsmn et l., 1977; Bginski nd Pierce, 1978; Collicutt nd Hochchk, 1977). In contrst, it hs een reported tht gint clms tht hrour symiotic zooxnthelle re cple of glutmine synthesis (Rees et l., 1994), nd tht resonly rpid glutmine synthesis nd turnover occurs in species tht excrete resonle mounts of purines (terrestril pulmonte snils nd some prosornch gstropods; see review y Bishop et l., 1983). Although GS hs een found in tissues of severl gstropod species (Cmpell nd Bishop, 197; Reddy nd Swmi, 1975; Horne, 1977), there hs een no report of the ctivity in tissues of nonsymiotic ivlves. Thus, this is the first report of the presence of glutmine synthetic cpcity in non-symiotic intertidl ivlve, nd therefore we mde n effort to elucidte its functionl role in P. expns (see elow). Since food ws withheld for 2 dys (3 dys prior to nd 17 dys during the experiment), the rte of proteolysis ws

6 461 K. C. Hiong nd others , Brckish wter Sewter Emersion Fig. 5. Effects of exposure to rckish wter (1, control), sewter (3 ) or ir (emersion) for 17 dys on the glutmte contents in the dductor muscle (white rs), foot muscle (grey rs), heptopncres (lck rs) nd mntle (htched rs) of Polymesod expns. Vlues re mens ± S.E.M. (N=4). Significntly different from the rckish wter vlue (P<.5); significntly different from the sewter vlue (P<.5). Brckish wter Sewter Emersion Fig. 6. Effects of exposure to rckish wter (1, control), sewter (3 ) or ir (emersion) for 17 dys on the glutmine contents in the dductor muscle (white rs), foot muscle (grey rs), heptopncres (lck rs) nd mntle (htched rs) of Polymesod expns. Vlues re mens ± S.E.M. (N=4). Significntly different from the rckish wter vlue (P<.5); significntly different from the sewter vlue (P<.5). likely to e greter thn the rte of protein synthesis, nd net protein degrdtion would hve occurred. Protein degrdtion together with incresed mino cid ctolism would led to n increse in the production of mmoni. Indeed, there ws significnt increse in the rte of mmoni excretion in clms mintined in rckish wter for 17 dys. These results indicte tht P. expns could effectively excrete the excess mmoni produced during this period of fsting in rckish wter. P. expns in sewter Exposure to sewter for 17 dys did not induce CPS III ctivity or ccumultion of ure in the heptopncres, mntle, dductor muscle nd foot muscle of P. expns. Thus, ure did not ct s n osmolyte in this mrsh clm. In invertertes, FAAs re mjor intrcellulr osmolytes, contriuting effectively to cell volume regultion (Pierce nd Greenerg, 1972). Concentrtions of intrcellulr FAAs of mrine or rckish wter ivlves fluctute in response to chnges in the mient slinity (Pierce, 1971; Giney, 1978,). In response to hypo-osmotic stress, intrcellulr levels of FAAs decrese s result of their efflux to extrcellulr comprtments (Pierce nd Greenerg, 1972, 1973) nd/or trnsformtion to other sustnces inside cells (Mtsushim et l., 1986). Conversely, in response to hyperosmotic stress, intrcellulr FAA concentrtions re elevted s result of protein degrdtion (Bginski nd Pierce, 1978; Livingstone et l., 1979; Henry et l., 198). Indeed, the TFAA content in the dductor muscle, foot muscle, heptopncres nd mntle incresed significntly y 2.6-, 2.3-, 4.2- nd 3.6-fold, respectively, in P. expns kept in sewter for 17 dys. These chnges were not result of chnges in wter contents of the tissues. Simultneously, the lnine content incresed y 3.1- to 6.4-fold, nd tht of glycine y 3.- to 5.9-fold. However, in terms of solute quntity, lnine ws the mjor contriutor, ecuse its percentge contriution to the TFAA pool incresed from % to % in vrious tissues. Thus, s suggested efore for other rckish nd mrine ivlves (Byne et l., 1976; Bishop et l., 1983), the functionl role of lnine in P. expns is minly connected with intrcellulr osmoregultion. The stedy-stte concentrtions of FAAs in tissues re determined nd mintined y the rtes of their degrdtion nd production (through proteolysis nd/or synthesis). Altertion of these two rtes would led to chnges in FAA concentrtion. The supply of FAAs for osmoregultory purposes through protein degrdtion in clms in high slinity is confirmed y studies on enzymes involved in mino cid metolism e.g. trnsminse (Greenwlt nd Bishop, 198) nd peptidse/proteinse (Byne et l., 1981; Deton et l., 1984). Since the mmoni excretion rtes in P. expns exposed to sewter were significntly greter thn in clms exposed to rckish wter on dy 15 nd dy 17, it cn e deduced tht the rtes of mino cid ctolism in the former were greter thn those in the ltter. In ddition, since there were greter TFAA contents in tissues of the former thn the ltter, it cn e concluded tht greter rte of protein

7 Glutmine synthesis in P. expns , Brckish wter Sewter Emersion Fig. 7. Effects of exposure to rckish wter (1, control), sewter (3 ) or ir (emersion) for 17 dys on the totl free mino cid (TFAA) contents in the dductor muscle (white rs), foot muscle (grey rs), heptopncres (lck rs) nd mntle (htched rs) of Polymesod expns. Vlues re mens ± S.E.M.(N=4). Significntly different from the rckish wter vlue (P<.5); significntly different from the sewter vlue (P<.5). degrdtion hd occurred in clms exposed to sewter compred with those exposed to rckish wter. Protein degrdtion leds to the relese of FAAs. FAAs cn e further ctolized, relesing mmoni. Increses in mmoni concentrtion would push rections ctlysed y glutmte dehydrogense nd lnine minotrnsferse towrds the synthesis of glutmte nd lnine (Newsholme nd Leech, 1983), leding to their ccumultion. Indeed, there were minor ut significnt increses in levels of glutmte in the heptopncres nd mntle of P. expns, indicting tht the glutmte dehydrogense rection hd een pertured. Furthermore, certin mino cids (e.g. rginine, glutmine, histidine nd proline) cn e converted to glutmte. Glutmte cn undergo trnsmintion with pyruvte, ctlyzed y lnine minotrnsferse, producing α- ketoglutrte without the relese of mmoni (Ip et l., 21,c; Chew et l., 23). α-ketoglutrte is then chnnelled into the tricroxylic cid (TCA) cycle for prtil metolism. The removl of mlte from the TCA cycle cn give continuous supply of pyruvte. Trnsmintion of pyruvte would produce lnine continuously, fcilitting the oxidtion of cron chins of certin mino cids without relesing mmoni. In contrst to lnine, there were no significnt increses in glutmine levels in vrious tissues of P. expns in sewter. As result, there were decreses in the percentges contriution of glutmine to the TFAA pools in the dductor muscle, foot muscle, heptopncres nd mntle of clms exposed to sewter (6., 9.1, 6.8 nd 5.5, respectively) Brckish wter Sewter Emersion Fig. 8. Effects of exposure to rckish wter (1, control), sewter (3 ) or ir (emersion) for 17 dys on the glutmine synthetse ctivity in the dductor muscle (white rs), foot muscle (grey rs), heptopncres (lck rs) nd mntle (htched rs) of Polymesod expns. Vlues re mens ± S.E.M. (N=4). Significntly different from the rckish wter vlue (P<.5); significntly different from the sewter vlue (P<.5). compred with those exposed to rckish wter (1.4, 14.7, 8.5 nd 16.1, respectively). Thus, it cn e concluded tht the physiologicl significnce of glutmine synthesis in P. expns is unrelted to osmoregultion. P. expns in ir Emersion inhiits severl metolic functions, such s feeding, respirtion nd excretion, in ivlves. In mny cses, oxygen consumption rtes re reduced during ir exposure. In response to lck of oxygen, the energy requirements of clms re stisfied through neroic glucose ctolism, for which lnine is one of the mjor end-products (de Zwn, 1977; Widdows et l., 1979; Henry et l., 198; Zururg nd de Zwn, 1981). However, P. expns ws exceptionl ecuse there were no significnt increses in lnine content in ny of the tissues studied fter 17 dys of eril exposure. This could e relted to the unique cpility of P. expns to mintin norml O 2 utiliztion rte during emersion (see Introduction). In spite of mintining eroic respirtion, the tissues of P. expns were not exposed to dehydrtion during emersion. Although P. expns does not encounter lck of oxygen in ir, it is confronted with prolems ssocited with mmoni excretion when wter is lcking. During eril exposure, mmoni cn e excreted into the mntle cvity fluid (Shick et l., 1988), ut mmoni concentrted therein would uild up nd led to ccumultion of mmoni in its tissues. Indeed, the mmoni contents in vrious tissues incresed etween 2.1- fold nd 3.-fold in P. expns exposed to ir for 17 dys. Becuse there ws no detectle CPS III ctivity nd no ccumultion of ure in vrious tissues of P. expns exposed

8 4612 K. C. Hiong nd others to ir for 17 dys, it cn e concluded tht, unlike in some gstropods (see review y Bishop et l., 1983), mmoni ccumulted during emersion ws not detoxified to ure in this mrsh clm. Since rections ctlyzed y trnsminses nd glutmte dehydrogense re ner equilirium in vivo (Newsholme nd Leech, 1983), sustrte concentrtions (e.g. mmoni) determine whether trnsmintion nd demintion of mino cids or mintion of α-keto cids occurs. An increse in tissue mmoni concentrtion would push the equilirium towrds mintion of α-keto cids through the rection ctlyzed y glutmte dehydrogense. This would theoreticlly result in n ccumultion of glutmte nd trnsminle non-essentil FAAs, including lnine (see ove), in the tissues. However, there were no increses in lnine content in ny of the tissues studied in P. expns exposed to ir for 17 dys. Hence, lnine ws not product mmoni detoxifiction in P. expns. Our results confirm tht mmoni ws detoxified to glutmine in P. expns during 17 dys of eril exposure. Tht mens glutmte formed from NH 4 + nd α-ketoglutrte rected further with mmoni, in the presence of ATP, to produce glutmine. The contents of glutmine in the dductor muscle, foot muscle, heptopncres nd mntle incresed 2.9-, 2.5-, 4.5- nd 3.4- fold, respectively. Simultneously, there were significnt increses in GS ctivities in the dductor muscle (1.56-fold) nd heptopncres (3.8-fold). This is the first report on the upregultion of GS nd ccumultion of glutmine in clm in response to eril exposure. Such phenomenon my e peculir to those ivlves tht cn mintin eroic energy metolism during emersion, ecuse synthesis of glutmine is ATP-dependent nd production of glutmte requires NAD(P)H. It would e energeticlly uneconomicl for n niml to increse glutmine nd glutmte syntheses while undergoing neroic energy metolism due to lck of oxygen supply, ecuse there would e decrese in ATP production nd prolems ssocited with redox lnce. Indeed, it hs een reported recently tht the swmp eel Monopterus lus does not ccumulte glutmine fter 4 dys of estivtion in hypoxic mud, ut ccumultes glutmine to high levels in its tissues during 6 dys of eril exposure (Chew et l., 25). Sokolowski et l. (23) determined FAA contents in the clm Mcom lthic L. from rckish wters of the southern Bltic Se, nd reported tht the overll temporl pttern of vritions in the concentrtion of glutmine in the period nlysed resemled, in generl, tht of lnine, with high vlues in the winter nd low in spring. Sokolowski et l. (23) suggested tht the physiologicl roles of these two mino cids were similr within sesonl cycle, despite the lck of evidence for such metolic connections. Contrry to the proposition of Sokilowski et l. (23), our results revel tht the functionl role of glutmine ws distinctly different from tht of lnine in P. expns. However, how lnine formtion nd glutmine synthesis, oth involving glutmte s sustrte, re regulted in P. expns in response to different environmentl conditions (exposure to sewter vs exposure to ir) is uncertin t present. Conclusion Returning to the initil impetus of this study, the results otined with P. expns demonstrte tht the doption of glutmine synthesis, similr to the doption of ure synthesis, for mmoni detoxifiction first took plce mong invertertes. Similr to other ivlve species, P. expns is incple of ure synthesis de novo, ut is cple of detoxifying mmoni to glutmine during emersion. The cpcity for glutmine synthesis ws present in vriety of tissues, including the heptopncres, mntle, dductor muscle nd foot muscle, in P. expns. This cpcity pprently extends to the lower vertertes, ecuse some ir-rething fishes re cple of synthesizing glutmine in their liver, muscles, nd digestive trcts s mjor strtegy to defend ginst mmoni toxicity during eril (Jow et l., 1999; Ip et l., 21; Chew et l., 21; Ty et l., 23) or environmentl (Peng et l., 1998; Anderson et l., 22; Ip et l., 24; Lim et l., 24) mmoni exposure. Brins of vertertes, from fish (Cmpell nd Anderson, 1991; Ip et l., 21, 24; Chew et l., 25) to mmmls (Cooper nd Plum, 1987; Brusilow, 22; Felipo nd Butterworth, 22), re cple of detoxifying mmoni to glutmine. However, higher vertertes such s mmmls do not dopt glutmine synthesis s mjor strtegy to del with mmoni toxicity in extr-crnil tissues, ecuse they hve evolved to depend minly on ure synthesis through the heptic OUC to detoxify mmoni (Cooper nd Plum, 1987). References Allen, K. (1961). The effect of slinity on the mino cid concentrtion in Rngi cunet (Pelecypod). Biol. Bull. 121, Anderson, P. M. nd Little, R. M. 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