Evidence for facilitated lactate uptake in lizard skeletal muscle

Transcription

1 The Journl of Experimentl Biology 24, (2) Printed in Gret Britin The Compny of Biologists Limited 2 JEB Evidence for fcilitted lctte uptke in lizrd skeletl muscle E. R. Donovn* nd T. T. Gleeson Section of Integrtive Physiology nd Neuroiology, E.P.O. Biology, University of Colordo Boulder, Boulder, CO , USA *e-mil: donovne@colordo.edu Accepted Septemer 2 To understnd more fully lctte metolism in reptilin muscle, lctte uptke in lizrd skeletl muscle ws mesured nd its similrities to the monocroxylte trnsport system found in mmmls were exmined. At 2 min, uptke rtes of 5 mmol l lctte into red iliofiulris (rif) were 2.4- nd 2.2-fold greter thn white iliofiulris (wif) nd mouse soleus, respectively. α- Cyno-4-hydroxycinnmte (5 mmol l ) cused little inhiition of uptke in wif ut cused % reduction in the uptke rte of lctte into rif, suggesting tht much of the lctte uptke y rif is vi protein-medited trnsport. N-ethymleimide (ETH) ( mmol l ) lso cused reduction in the rte of uptke, ut mesurements of denylte nd phosphocretine Summry concentrtions show tht ETH hd serious effects on rif nd wif nd my not e pproprite for trnsport inhiition studies in reptiles. The higher net uptke rte y rif thn y wif grees with the fct tht rif shows much higher rtes of lctte utiliztion nd incorportion into glycogen thn wif. This study lso suggests tht lctte uptke y reptilin muscle is similr to tht y mmmlin muscle nd tht, evolutionrily, this trnsport system my e reltively conserved even in nimls with very different ptterns of lctte metolism. Key words: monocroxylte trnsporter, lizrd, Dipsosurus dorslis, muscle fire type, lctic cid. Introduction Lctte plys numerous roles in skeletl muscle. It is metolic end-product of glycolysis, mjor source of energy for oxidtive fiers, mitochondril redox shuttle compound nd sustrte for replenishment of glycogen stores (Brooks, 99; Duouchud et l., 2; Gldden, 2). The chnges in ph concurrent with the uild-up of lctte in the muscle nd lood re ssocited with ftigue of the muscle nd inhiition of the metolic pthwys required for continued muscle function nd recovery fter ctivity (Juel, 996; Roth, 99). In the light of the role of lctte s oth end-product nd sustrte, nd ecuse of the effects of lctte ccumultion, study of its metolism nd trnsport is importnt to understnd how it ffects the ctivity physiology of nimls. Reserch on lctte production nd removl during ctivity nd recovery hs shown different ptterns for reptiles nd mmmls (Gleeson, 996). In mmmls, lctte produced during ctivity is primrily oxidized in muscle nd is removed reltively quickly during recovery (Brooks nd Gesser, 98). Brooks hs descried system in which lctte produced y some fier types is exported to nd oxidtively removed y other fier types (Brooks, 986). There is lso some evidence tht glycogenic removl of lctte occurs in rodent muscle following intense, rief exercise (Nikolovski et l., 996). Reptiles tend to rely upon glycolytic pthwys to support most of their vigorous ctivity nd, s result, they cn ccumulte more lctte, nd t fster rte, thn mmmls (Gleeson, 996). During recovery, much of this lctte is used for glycogenesis to restore muscle glycogen levels, nd little is oxidized (Gleeson, 99). These ptterns of metolism imply need for some sort of trnsport system for the removl of lctte from sites of production nd uptke t sites of metolism. Studies in vrious mmmlin tissues hve shown tht lctte cn e trnsported cross the cell memrne y three mechnisms: y free diffusion, y monocroxylte trnsporter (MCT), which works s lctte/h + symporter, or y nionic ntiports such s lctte/cl -HCO 3 exchngers (Deutick et l., 982; Poole nd Hlestrp, 993). The ltter pthwy does not seem to ply significnt role in mmmlin skeletl muscle (Juel nd Wirnd, 989; Roth nd Brooks, 99; McDermott nd Bonen, 994). The undissocited cid cn esily diffuse cross the memrne (Poole nd Hlestrp, 993) ut, t physiologicl ph, more thn 95 % of the lctic cid is dissocited s lctte nion nd proton (pk=3.86). It ppers tht the mjority of lctte trnsport in mmmls is due to MCT-protein-medited trnsport (Juel nd Wirnd, 989; Roth nd Brooks, 99,; Bonen nd McCullgh, 994; McDermott nd Bonen, 994). The MCT proteins trnsport short-chin monocroxyltes, such s lctte nd pyruvte, nd re stereospecific for L-lctte

2 4 E. R. Donovn nd T. T. Gleeson over D-lctte. They hve een shown to e inhiited oth y non-specific protein modifiers nd y non-trnsportle monocroxylte nlogs (Juel nd Wirnd, 989; Roth nd Brooks, 99; Poole nd Hlestrp, 993; McDermott nd Bonen, 994). A numer of different isoforms of the protein hve een identified, with specific isoforms eing ssocited with different tissues (Hlestrp nd Price, 999). MCT content correltes with muscle s oxidtive cpcity, while MCT4 is expressed more in glycolytic fiers (McCullgh et l., 996; Pilegrd et l., 999; Hlestrp nd Price, 999; Bonen et l., 2). Despite the work done on mmmls, only smll numer of studies hve investigted the trnsport of lctte in other nimls. A study of crustcens suggests lck of ny fcilitted lctte trnsport (Kinsey nd Ellington, 996). Studies of lctte efflux in rinow trout muscle seem to show trnsport similr to tht oserved in mmmls, ut studies of uptke indicte some significnt differences in responses to inhiitors nd suggest the possiility of ctive trnsport, with lctte eing tken up ginst its electrochemicl grdient (Wng et l., 997; Leree nd Millign, 999). These processes hve yet to e exmined in reptiles, nd their study will help develop our understnding of the evolutionry history of lctte trnsport. Becuse of the high degree of dependence on glycolytic metolism for ctivity of mny reptiles (Bennett, 978), knowledge of the mechnism of lctte trnsport in reptilin skeletl muscle will lso e key to understnding the physiologicl limittions on the ctivity cpilities of this clss of nimls. The gol of this study ws to exmine the reptilin lctte trnsport system in the context of the recovery metolism of the desert igun Dipsosurus dorslis. This study ttempted to descrie lctte uptke in oth oxidtive nd glycolytic skeletl muscle fiers of D. dorslis. The responses of these muscles to vrious inhiitors tht hve een shown to inhiit the MCT proteins of mmmls were mesured to determine whether they hve lctte trnsport systems similr to those of mmmls. Mterils nd methods Animls cre/tissue preprtion Fifteen desert iguns (Dipsosurus dorslis) were collected ner Cthedrl City, CA, USA, on permit from Cliforni Fish nd Gme nd housed in sndy cges with 24-h ccess to het lmp nd mple hiding plces; they were segregted y sex. They were fed diet of lettuce mixed with powdered rt chow three times weekly. Lizrds were killed y decpittion. The iliofiulris muscle (IF) ws removed nd plced in lizrd Ringer s preprtion solution (Guillette, 982), with finl concentrtions of solutes (in mmol l ) s follows: NCl, 57.; KCl, 4.6; NHCO 3, 2.; CCl 2,.45; NH 2 PO 4.H 2 O, 2.6; MgSO 4,.. The solution ws uled with 95 % O 2 nd 5% CO 2. The IF hs distinct red nd white sections (rif nd wif, respectively), which re composed primrily of oxidtive nd glycolytic fiers, respectively (Putnm et l., 98). These sections were seprted from ech other, further divided into two (rif) or three (wif) undles nd clened of dmged fiers under 7.5 mgnifiction. Ech undle ws pinned t resting length through its tendons to Tygon mounts. Five mice (Mus domesticus) from n outred stock (CF-) were otined from Ssco Inc., housed in common cge on 2 h:2 h light:drk cycle nd given food nd wter d liitum. The mice were killed y cervicl disloction. The soleus muscle (SOL) ws removed from the lim nd plced in Ringer s preprtion solution (Dwson et l., 998) uled with 95 % O 2 nd 5 % CO 2. The muscle ws seprted into two fier undles, which were then clened of dmged fiers nd mounted. All procedures were pproved y the niml cre nd use committee of the University of Colordo. Incution medi Rdiochemicls were otined from ICN Phrmceuticls (L-[U- 4 C]lctic cid nd D-[U- 4 C]lctic cid) nd NEN (sucrose[fructose-- 3 H(N)]). All other chemicls were purchsed from Sigm Chemicl. All solutions descried elow were mde using the Ringer s (either mouse or lizrd, s pproprite) solutions descried for use in the tissue preprtion ove, plus dditionl chemicls s indicted elow. All the solutions were equilirted with 95 % O 2 nd 5% CO 2. After mounting, muscle undles were plced for 3 min in pre-incution solution with 5 mmol l lctte to estlish n intrcellulr concentrtion in ll the fier undles similr to tht found in resting D. dorslis muscle (Crocker et l., 2). The pre-incution solution lso contined no inhiitor (control), 5 mmol l α-cyno-4-hydroxycinnmte (CIN) or mmol l N-ethymleimide (ETH). A decrese in the rte of lctte uptke in the presence of ETH, which is generl sulfhydryl modifier, nd CIN, which cts s non-trnsportle monocroxylte nlog, is frequently used s n indictor of protein-medited trnsport (Wtt et l., 988; Roth nd Brooks, 99; McDermott nd Bonen, 994; Leree nd Millign, 999). Both ETH nd CIN were dissolved directly in the Ringer s incution solution. The solutions for ll incutions were t 37 C nd ph 7.4 for mouse muscle fier undles nd 4 C nd ph 7.2 for lizrd muscle fier undles. After pre-incution, muscle fier undles were used in one of three seprte procedures. The first two tested how incution time nd externl lctte concentrtion ffected estimtes of rtes of lctte uptke. In the first procedure, muscle undles were moved from the control pre-incution solution into 5 mmol l lctte incution solution contining 8.5 kbq of [ 4 C]lctte nd 37 kbq of [ 3 H]sucrose nd were incuted for 5 min. In the second procedure, undles were incuted with rdioleled sucrose nd either L- or D-lctte s ove for 2 min in L- or D-lctte concentrtions of 5 mmol l. To mintin the sme osmolrity in ll incutions nd prevent ny effects of osmotic chnge, these solutions hd their NCl concentrtions decresed in proportion to the mount of sodium lctte dded. The third procedure determined the effects of different lctte

3 Lctte uptke in lizrd muscle 4 concentrtions nd different inhiitors on lctte uptke in lizrd muscle. In this procedure, undles were moved from the pre-incution solution into solutions contining either 5 or 5 mmol l lctte, [ 4 C]lctte, [ 3 H]sucrose nd either the sme inhiitor s in their pre-incution or non-inhiitor control. These incutions were crried out for 2 min. After ech incution, muscle undles were immeditely lotted, frozen in liquid N 2 nd stored t 7 C until nlyzed (usully within 3 dys). A smple of ech incution solution ws lso frozen for lter nlysis. Lctte uptke mesurement Pre-weighed frozen muscle smples were dropped into.3 ml of hot mol l NOH, vortexed for 5 s nd plced in oiling wter th for 2 min. This solution ws neutrlized with.3 ml of mol l HCl nd llowed to cool for 2 min. To mesure lctte uptke,.4 ml of the neutrlized solution ws dded to 3 ml of Scintiverse II scintilltion cocktil. The incution medium ws prepred for nlysis y dding.3 ml to 3 ml of Scintiverse cocktil. An LKB Wllc, model 24 liquid scintilltion counter, which hs integrted softwre cple of counting in multiple emission spectrum windows, ws used to mesure 3 H nd 4 C rdioctivity concurrently in ech smple. Lctte uptke ws clculted from 4 C uptke, using the [ 3 H]sucrose s n extrcellulr spce mrker to correct for the 4 C in the extrcellulr spce (Juel nd Wirnd, 989). The following eqution ws used to determine [ 4 C]lctte uptke in units of disintegrtions per minute (disints min ): [ 4 C]lctte uptke = [ 4 C]lctte m ([ 4 C]lctte inc /[ 3 H]sucrose inc )([ 3 H]sucrose m ), () where the suscripts m nd inc indicte totl disints min for muscle nd incution solution, respectively. The disints min vlues were then converted into nmoles of lctte using the specific ctivity of lctte in the incution solution. Metolite ssys Muscle smples were nlyzed for denyltes nd phosphgens using high-performnce liquid chromtogrphy (HPLC), t rest, immeditely fter dissection nd fter ech of the incution tretments. Resting smples were otined y freeze-clmping the entire leg immeditely fter decpittion nd then dissecting out the frozen muscle. Post-dissection smples were otined y freezing the muscle fier undles in liquid N 2 immeditely fter they hd een mounted on the Tygon strip. Frozen sections of ech muscle were ground in -fold dilution of 6 % ice-cold HClO 4 nd then neutrlized with 5 mol l K 2 CO 3. This solution ws centrifuged for 5 min, nd the superntnt ws decnted nd centrifuged for 6 min t revs min, frozen, thwed nd centrifuged gin for 6 min t revs min. This lst superntnt ws nd filtered through Rinin.45 µm filter. Superntnt ( ml) ws injected into moile phse consisting of 65 mmol l KH 2 PO 4 nd cetonitrile (:2.), t ph of 6.9, which flowed through Microsor MV-A mino propyl column t rte of. ml min. The effluent ws nlyzed with Rinin Dynmx spectrophotometer (model UV-C) t 245 nm for AMP, ADP nd ATP in series with Millipore Lmd Mx spectrophotometer (model 48) t 25 nm for cretine nd phosphocretine. Stndrds were prepred nd nlyzed in the sme mnner s smples, nd their metolite concentrtions did not chnge significntly with multiple freeze/thw cycles. Lctte concentrtions in the muscles fter the 3 min preincution were mesured spectrophotometriclly (Gleeson, 985). Adenylte energy chrge (AEC) ws clculted s: AEC = ([ATP] + G[ADP]) / ([ATP] + [ADP] + [AMP]). (2) Sttisticl nlyses The results were nlyzed using the progrm SttView ver. 5.. (SAS Institute Inc.). Multiple regression nlysis ws used to test the effects of incution time, externl lctte concentrtion nd muscle chrcteristics on lctte uptke. A one-group t-test ws used to determine whether the intrcellulr lctte concentrtion ws significntly different from 5 mmol l fter the pre-incution. Anlysis of vrince (ANOVA) with Fisher s PLSD nlysis ws used to compre the following; rif, wif nd SOL uptke rtes t 2 min, control lctte uptke rtes etween, nd the inhiitor effects within, the 5 nd 5 mmol l lctte incutions, nd the AEC nd cretine, phosphocretine concentrtions throughout the procedure. All rtes nd concentrtions reported re mens ± S.E.M. All effects were considered significnt t P.5. Results Neither the mss of the niml nor the dimeter or mss of the muscle fier undle (Tle ) ws found to hve ny significnt correltion to the rtes of lctte uptke in the time- Tle. Dt for the nimls nd muscle undles used in incutions Men S.E.M. N Animl mss (mg) Mouse Lizrd Muscle mss (mg) Soleus Red IF White IF Muscle dimeter (mm) Soleus.2. 3 Red IF White IF IF, iliofiulrs.

4 42 E. R. Donovn nd T. T. Gleeson Rte of lctte uptke (nmol mg - muscle min - ) Incution time (min) Fig.. Men rte of lctte uptke y mouse soleus muscle during 5 min incutions. Vlues re mens ± S.E.M., N=3 5, except t min where N=2. At some points error rs re smller thn the symols. course experiments for D. dorslis (prtil regression coefficient P-vlues: for niml mss, P=.35; for undle mss, P=.93; for undle dimeter, P=.77). As result, these vriles were not included in susequent nlyses of the effects of inhiitors on the rte of lctte uptke. Ech muscle undle ws considered to e n independent smple ecuse of the 3 min pre-incution tretment. Pre-incution of undles in 5 mmol l lctte estlished n intrcellulr lctte concentrtion of 4.7±.2 mmol l (N=), which ws not significntly different from 5 mmol l (P=.5). To test the efficcy of our mesurements of reptilin lctte uptke, uptke in mouse SOL ws mesured for comprison with vlues from other studies. The uptke rte of lctte y mouse SOL (Fig. ) decresed significntly over time (P=.3) nd ppered to stilize t.26±.5 nmol mg muscle min y min of incution. Anlysis showed tht the decrese ws liner (P=. for the time time interction). The uptke rte of lctte y the lizrd IF (Fig. 2) decresed s function of time (P<.). Across ll incution times, lctte uptke y rif ws higher thn tht y wif (P<.), ut uptke rtes for rif nd wif were not ffected differently y incresed incution time (P=.6 for the time fier interction). At 2 min, the uptke y rif ws higher thn the rtes of oth wif (2.4- fold, P=.) nd SOL (2.2-fold, P=.9), while the rtes of uptke y wif nd SOL did not differ (P=.87). To estimte the uptke rte of the whole IF, the uptke rtes of the rif nd wif were multiplied y their percentge contriution to the whole IF nd summed. The muscle composition ws estimted s 7. % wif nd 28.9 % rif (Putnm et l., 98). At 2 min, the uptke rte of the whole IF ws pproximtely.5 times fster thn tht of the SOL muscle. The effects of lctte concentrtion on uptke Rte of lctte uptke (nmol mg - muscle min - ) Rte of lctte uptke (nmol mg - muscle min - ) Incution time (min) Fig. 2. Men rte of lctte uptke y Dipsosurus dorslis red iliofiulris (rif) ( ) nd white iliofiulris (wif) ( ) during 5 min incutions. Vlues re mens ± S.E.M., N=3 4 for rif nd wif t ech incution time, except t 5 min where N=2. At some points error rs re smller thn the symols. mesurements re shown in Fig. 3 nd Tle 2. A multiple regression nlysis of the dt for L-lctte uptke show tht, for rif, there ws significnt effect of lctte concentrtion on the rte of lctte uptke (P<.) nd tht this effect decresed s lctte concentrtion incresed (P=.24 for the [lctte] [lctte] interction), suggesting sturle trnsport process. For wif, incresing L-lctte concentrtion cused liner increse in uptke rtes (P=.9). WIF showed no sturtion kinetics (P=.88 for the [lctte] [lctte] [Lctte] (mmol l - ) Fig. 3. Men rte of lctte uptke for Dipsosurus dorslis red iliofiulris (rif) ( ) nd white iliofiulris (wif) ( ) during 2 min incutions with 5 mmol l intrcellulr nd 5 mmol l extrcellulr lctte. The lines re the diffusion-corrected uptke rtes for rif (dshed line) nd wif (solid line). Vlues re mens ± S.E.M., N=4 6 for rif nd N=6 for wif. At some points error rs re smller thn the symols.

5 Lctte uptke in lizrd muscle 43 Tle 2. Rtes of uptke of L-lctte nd D-lctte Uptke rtes (nmol mg Concentrtion min ) (mmol l ) L-Lctte D-Lctte rif 5.36±.8.5±. 5.4±..3± ±.5.26± ±.6.46±. 3.89±.36.3±.3 wif 5.8±.4.2±. 5.38±.5.35± ±.6.56±.3 6.3±.8.39± ±. 2.32±.55 Vlues re mens ± S.E.M., N 4. rif, red iliofiulris; wif, white iliofiulris. interction). The uptke rtes of D-lctte were liner for oth rif nd wif nd were significntly lower thn the corresponding uptke rtes for L-lctte (P=.2 for rif nd P=.25 for wif) (Tle 2). These dt were used to correct the L-lctte uptke rtes for diffusion, nd the resulting curves (Fig. 3) were fitted to Linewever Burke plot. These plots gve n pprent K m of mmol l nd V mx of 4.6 nmol mg muscle min for rif nd K m of 3.44 mmol l nd V mx of.94 nmol mg muscle min for wif. The lctte uptke rtes for rif nd wif were.85 nd.28 nmol mg muscle min for the 5 mmol l incution nd.36 nd.8 nmol mg muscle min for the 5 mmol l incutions. The 5 mmol l incution uptke rtes were significntly greter thn the 5 mmol l rtes for rif (P=.4) ut not for wif (P=.98). Anlysis of control lctte uptke rtes in the inhiition experiments showed tht rif hd higher uptke rte thn wif in oth the 5 nd 5 mmol l lctte incution conditions (P<.) (Fig. 4A,B). In the 5 mmol l lctte incutions, CIN significntly decresed uptke reltive to the uninhiited control in rif y.35 nmol mg muscle min (P<.) ut not in wif (decrese of.7 nmol mg muscle min, P=.5). ETH lso significntly decresed uptke in rif y.56 nmol mg muscle min (P<.) ut not in wif (decrese of.5 nmol mg muscle min, P=.27). In the 5 mmol l incutions, CIN nd ETH significntly decresed the uptke rte y rif (decrese of.9 nd.28 nmol mg muscle min, respectively; P<.5) nd wif (decrese of. nd.8 nmol mg muscle min ; P=.7 nd P=.38, respectively). The denylte energy chrge decresed significntly in oth the rif nd wif ETH incutions (P<.) ut there were no significnt decreses t ny other point in the experiment (Tle 3). Cretine concentrtions dropped significntly in ll cses except in the rif ETH incutions, wheres Rte of lctte uptke (nmol mg - muscle min - ) CON CIN ETH phosphocretine concentrtion decresed only in the wif ETH incution (P=.6). Discussion The findings of this study show tht fcilitted trnsport is present in the skeletl muscles of D. dorslis nd tht this process plys n importnt role in trnsporting lctte t concentrtions seen during typicl ctivities. Lctte metolism hs een shown to ply n importnt prt in the physiology nd energetics of lizrds, nd there hs een much study of lctte production, its removl nd its metolic fte in different tissues. Mechnisms of lctte trnsport to nd CON CIN ETH Incution condition Fig. 4. Rtes of lctte uptke in (A) 5 mmol l intrcellulr:5 mmol l extrcellulr lctte concentrtion grdient nd (B) 5 mmol l intrcellulr:5 mmol l extrcellulr concentrtion grdient in the presence of the monocroxylte trnsporter inhiitors N-ethymleimide (ETH) nd α-cyno-4- hydroxycinnmte (CIN). Vlues re mens ± S.E.M. for red iliofiulris (rif) (htched columns) nd white iliofiulris (wif) (open columns); N=4 8 in A nd N=3 6 in B for ech fier type in ech incution condition. Comprisons were mde only within [lctte] conditions nd within fier types. Columns with different superscripts were significntly different (P.5) for sttisticl comprisons mde etween inhiitor conditions, within muscle type. CON, control. c A B

6 44 E. R. Donovn nd T. T. Gleeson Tle 3. Metolite concentrtions nd denylte energy chrge in red nd white iliofiulris muscle throughout the uptke inhiition experiment Post- Control CIN ETH In vivo resting dissection incution incution incution Metolite (mmol l ) (mmol l ) (mmol l ) (mmol l ) (mmol l ) rif AEC.92±.2.9±.2.87±.4.92±..2±.6* ATP 5.8±.9 3.5± ± ±.9.7±.8* ADP.33±..5±.5.22±.7.3±.8.6±.4 AMP.24±.3.24±.6.24±.5.9±.3.4±.3 PCr 22.69± ± ± ± ±7.2 Cr 8.4± ±2.53* 3.57±.57* 3.63±.27* 8.8±3.5* wif AEC.92±.3.9±.3.9±.2.9±.3.67±.4* ATP 8.89± ± ± ±.47.3±.* ADP.32±.2.33±.8.9±.5.4±.7.±. AMP.55±.4.5±..37±.9.35±.6*.5±. PCr 37.34± ± ± ± ±3.7* Cr 23.9± ±.58* 5.4±.8* 7.9±.6* 4.5±.4* Vlues re mens ± S.E.M., N=3 (except for rest, N=5). AEC, denylte energy chrge; Cr, cretine; PCr, phosphocretine; rif, red iliofiulris; wif, white iliofiulris. *Indictes significnt difference from the resting vlue. from these res of production nd utiliztion hve not yet een exmined. Our demonstrtion of ctive trnsport of lctte opens new re of study into the ctivity physiology of lizrds nd the fctors tht regulte their musculr function. Assessment of uptke procedure To ssess the overll effects of the incution procedure nd inhiitors on muscle condition, AEC nd cretine nd phosphocretine concentrtions were mesured t vrious points in the 5 mmol l inhiition procedure. These results (Tle 3) show tht, except for the muscles in the ETH incutions, ll the muscle fier undles ppered to mintin n energy chrge close to resting levels throughout the procedure nd t the end of the incutions. However, the diminished PCr concentrtions nd AEC of the ETH-inhiited muscles suggest pthologicl effects on the physiology of the muscle cells eyond simple inhiition of lctte trnsport. This rises questions out whether ETH is n pproprite inhiitor for studying protein-medited trnsport in reptilin muscle fiers. Our results for the ETH incutions should not, therefore, e used to drw ny conclusions out the processes of lctte trnsport in the muscles exmined ecuse they my not e n indiction of the ctul sensitivity of reptilin lctte trnsport proteins to ETH inhiition nd insted simply e the result of deleterious effects of ETH on reptilin cellulr homeostsis. The difference etween resting nd experimentl condition cretine concentrtions merits comment. Phosphocretine levels remined elevted; however, cretine levels were low reltive to resting vlues (Tle 3). Cretine loss ws not ssocited with similr decline in the concentrtion of ny denylte. Lower cretine concentrtions in incuted muscle do not pper to e due to loss of memrne integrity ecuse sucrose prtitioning persisted in these tissues. Experiments involving electricl stimultion in which the IF ws sujected to identicl dissection nd similr hndling showed tht the muscles remined cple of norml contrctions for severl hours (T. T. Gleeson, unpulished dt) further suggesting tht memrne viility remined throughout our incution procedure. Cretine production nd rekdown hve not een well studied in lizrds, ut in mmmls they hve een shown to involve multiple steps, some reversile nd some not, which eventully led to the formtion of cretinine (Wyss nd Kddurh-Douk, 2). It is possile tht some of the degrdtive steps proceed t greter rte in lizrds thn in mmmls, leding to greter loss over time. Mesurements of lctte uptke in the mouse SOL were primrily to provide n ssessment of our procedure. Our results for these mesurements (Fig. ), while somewht lower thn vlues reported elsewhere for similr incution conditions (Bonen nd McCullgh, 994; Juel nd Wirnd, 989; McDermott nd Bonen, 994), re of the sme mgnitude nd show tht our methods were sound. Time- nd concentrtion-dependency of lctte uptke Mesurements of lctte uptke y rif over time show pttern similr to tht in mmmls. Initil rtes were rpid nd decresed linerly for the first 3 min efore leveling off (Fig. 2). Fig. 3 shows oth the totl nd diffusion-corrected lctte uptke rtes. At physiologicl concentrtions, it ppers tht pproximtely % of the trnsport is vi diffusion in oth rif nd wif, while t higher concentrtions ( mmol l ) nerly 6 % of the trnsport is due to

7 Lctte uptke in lizrd muscle 45 diffusion. The K m nd V mx mesured for the rif re much higher thn vlues reported for mmmlin skeletl muscle, suggesting high-cpcity, low-ffinity trnsport system reltive to mmmlin muscle (Wtt et l., 988; Juel nd Wirnd, 989; McDermott nd Bonen, 994). Physiologicl lctte concentrtions in reptiles re often higher thn in mmmls (Gleeson, 996), nd these greter trnsport cpcities could e eneficil in iding recovery fter ctivity. Inhiitor effects CIN (5 mmol l ) hs een shown to inhiit uptke of mmol l lctte into isolted mouse SOL (McDermott nd Bonen, 994) nd perfused rt hindlim (Wtt et l., 988) y 22 %. Our dt show inhiition of lctte uptke into rif y 5 mmol l CIN of 54 % nd 42 % in the 5 nd 5 mmol l lctte tretments, respectively, nd of 54 % for wif in the 5 mmol l lctte tretment (Fig. 4A,B). The fct tht the inhiition mesured here is greter thn hs een recorded in mmmlin muscle my indicte either tht the oxidtive skeletl muscle of D. dorslis hs higher percentge of its lctte uptke vi protein-medited system or tht its proteins simply hve higher ffinity for these inhiitors. The significnt inhiition of uptke into rif fiers y CIN in oth 5 nd 5 mmol l lctte incutions suggests tht fcilitted trnsport plys mjor role in the uptke of lctte t oth resting nd post-ctivity lctte concentrtions. The significnt inhiition of uptke into wif y CIN in the 5 mmol l lctte incution nd the pprent sturtion kinetics of the diffusion-corrected lctte uptke suggest tht MCT-like fcilitted trnsport my lso ply role in lctte uptke into these fiers. However, there ws no significnt inhiition of the uptke rtes into wif for the 5 mmol l incutions, nd L-lctte uptke showed no sturtion up to mmol l. This could indicte tht fcilitted trnsport my ply mjor role in lctte uptke in wif under resting conditions ut tht it my ply lesser role in steeper lctte concentrtion grdients. The low K m nd V mx otined for wif, reltive to rif, support this possiility. To the est of our knowledge, the effects of CIN on mmmlin white muscle hve yet to e ddressed. There re dt on the effect of CIN on lctte trnsport in trout white muscle suggesting tht CIN inhiits lctte efflux ut not uptke. These dt suggest tht different pthwys of proteinmedited uptke nd efflux re present in trout. Trout, however, show pttern of ctive lctte retention in white muscle tht is very different from the pttern of rpid relese into the loodstrem seen in reptilin nd mmmlin white muscle nd tht mkes comprisons of CIN inhiition etween trout nd other tx difficult (Leree nd Millign, 999; Wng et l., 997). Tken together, the inhiition y CIN t 5 mmol l nd the diffusion-corrected sturtion kinetic dt suggest tht lizrd white muscle utilizes MCT-medited trnsport for uptke ut not to the sme degree s ppers to e the cse for rif. While MCT trnsport my ply limited role in uptke into wif, this does not rule out the presence of MCT-fcilitted lctte efflux in lizrd glycolytic muscle. In mmmlin muscle, there is evidence tht lctte uptke nd efflux tend to occur vi different MCT isoforms (MCT nd MCT4, respectively) which show different distriutions in oxidtive nd glycolytic muscles (McCullgh et l., 996; Wilson et l., 998; Bonen et l., 2). If this is lso the cse in lizrds, it would e necessry to exmine the effects of trnsport inhiitors on lctte efflux from wif to ssess more fully the role of MCT trnsport in this muscle. Correltion etween uptke rtes nd lctte production/utiliztion rtes Post-exercise lctte concentrtions in D. dorslis rif nd wif cn e rised to 34.5 nd 47.9 mmol kg, respectively (Gleeson nd Dlessio, 99). Deling with this lctte is vitl oth for cid se lnce nd for recovery of depleted glycogen stores. After 2 h of recovery from exhustive ctivity, incorportion of lctte into glycogen in vivo is pproximtely 48 nmol kg in rif nd 8 nmol kg in wif (Gleeson nd Dlessio, 99), nd skeletl muscle glycogen synthesis ccounts for pproximtely 5 % of the lctte removl during this period (Gleeson nd Dllesio, 989). The high rtes of lctte uptke mesured for rif nd the lower rtes for wif relte well to their respective rtes of lctte utiliztion for glycogen synthesis. Glycolytic muscle is the mjor site of lctte production, nd the intrcellulr concentrtions post-ctivity re likely to e high enough to support the low rtes of glycogen synthesis of these fiers during recovery. Although there ppers to e little fcilitted lctte uptke, there my e lctte efflux from these fiers t significnt rte during recovery to speed the restortion of intrcellulr ph nd mke lctte ville to the oxidtive fiers for metolism. Agin, mesurements of lctte efflux would e required to determine ny role of MCT proteins in this process. Comprison of rif nd wif with oxidtive nd glycolytic muscles in other nimls Numerous studies hve shown tht mmmlin glycolytic muscles hve lower rtes of lctte uptke thn oxidtive muscles (Juel et l., 99; Bonen nd McCullgh, 994; McCullgh et l., 997), nd our results for reptilin muscle re similr. Across rnge of rt nd mouse skeletl muscles, there seems to e strong correltion etween the rtes of lctte uptke nd the oxidtive cpcity of the muscles (McCullgh et l., 996). On the sis of citrte synthse ctivities, lizrd rif hs n oxidtive cpcity similr to or lower thn tht of these mmmlin oxidtive muscles (Gleeson nd Hrrison, 988; Moerlnd nd Kushmerick, 994; McCullgh et l., 996). Therefore, lizrd rif might lso e expected to hve the sme or lower uptke rte. Our results indicte tht rif hs significntly higher lctte uptke rte thn the mouse SOL (Figs, 2), suggesting tht the correltion seen etween oxidtive cpcity nd lctte uptke in mmmls is not present in D. dorslis.

8 46 E. R. Donovn nd T. T. Gleeson Concluding remrks This study demonstrtes tht protein-medited lctte trnsport system functions in reptilin skeletl muscle. It is the first report of such trnsport in this verterte clss. Lctte uptke rtes re higher in oxidtive thn in glycolytic muscle, nd lctte uptke is more sensitive to inhiitors in oxidtive muscle. The dt presented here lso suggest tht rtes of uptke my e higher in reptilin oxidtive muscle thn they re in mmmlin muscle of equivlent, or higher, oxidtive cpcity. The uptke my e due to similr group of MCT proteins to tht descried in mmmlin muscle. We would like to thnk Dr Crlos Crocker for his help with the HPLC dt collection nd nlysis. This work ws supported y NSF grnt References Bennett, A. F. (978). Activity metolism of the lower vertertes. Annu. Rev. Physiol. 4, Bonen, A. nd McCullgh, K. J. A. (994). Effects of exercise on lctte trnsport into mouse skeletl muscles. Cn. J. Appl. Physiol. 9, Bonen, A., Miskovic, D., Tonouchi, M., Lemieux, K., Wilson, M. C., Mrette, A. nd Hlestrp, A. P. (2). Aundnce nd sucellulr distriution of MCT nd MCT4 in hert nd fst-twitch skeletl muscles. Am. J. Physiol. 278, E67 E77. Brooks, G. A. (986). The lctte shuttle during exercise nd recovery. Med. Sci. Sports Exerc. 8, Brooks, G. A. (99). Current concepts in lctte exchnge. Med. Sci. Sports Exerc. 23, Brooks, G. A. nd Gesser, G. A. (98). End points of lctte nd glucose metolism fter exhustive exercise. J. Appl. Physiol. 49, Crocker, C. E., Hncock, T. V. nd Gleeson, T. T. (2). Post-exercise metolite concentrtions in skeletl muscle from the desert igun, Dipsosurus dorslis. Physiologist 43, 356. Dwson, R. M. C., Elliot, D. C., Elliot, W. H. nd Jones, K. M. (998). Physiologicl medi. In Dt for Biochemicl Reserch, third edition (ed. R. M. C. Dwson), pp New York: Oxford University Press. Deuticke, B., Beyer, E. nd Forst, B. (982). Discrimintion of three prllel pthwys of lctte trnsport in the humn erythrocyte memrne y inhiitors nd kinetic properties. Biochim. Biophys. Act 684, 96. Duouchud, H., Butterfield, G. E., Wolfel, E. E., Bergmn, B. C. nd Brooks, G. A. (2). Endurnce trining, expression nd physiology of LDH, MCT nd MCT4 in humn skeletl muscle. Am. J. Physiol. 278, E57 E579. Gldden, B. L. (2). Lctic cid: New roles in new millennium. Proc. Ntl. Acd. Sci. USA 98, Gleeson, T. T. (985). Glycogen synthesis from lctte in skeletl muscle of the lizrd Dipsosurus dorslis. J. Comp. Physiol. B 56, Gleeson, T. T. (99). Ptterns of metolic recovery from exercise in mphiins nd reptiles. J. Exp. Biol. 6, Gleeson, T. T. (996). Post-exercise lctte metolism: comprtive review of sites, pthwys nd regultion. Annu. Rev. Physiol. 58, Gleeson, T. T. nd Dlessio, P. M. (989). Lctte nd glycogen metolism in the lizrd Dipsosurus dorslis following exhustive exercise. J. Exp. Biol. 44, Gleeson, T. T. nd Dlessio, P. M. (99). Lctte: sustrte for reptilin muscle gluconeogenesis following exhustive exercise. J. Comp. Physiol. B 6, Gleeson, T. T. nd Hrrison, J. M. (988). Muscle composition nd its reltion to sprint running in the lizrd Dipsosurus dorslis. Am. J. Physiol. 255, R47 R477. Guillette, L. J., Jr (982). A physiologicl (Ringer s) solution for noline lizrds. Herpetol. Rev. 3, Hlestrp, A. P. nd Price, N. T. (999). The proton-linked monocroxylte trnsporter (MCT) fmily: structure, function nd regultion. Biochem. J. 343, Juel, C. (996). Lctte/proton co-trnsport in skeletl muscle: regultion nd importnce for ph homeostsis. Act Physiol. Scnd. 56, Juel, C., Honig, A. nd Pilegrd, H. (99). Muscle lctte trnsport studied in srcolemml gint vesicles: dependence on fier type nd ge. Act Physiol. Scnd. 43, Juel, C. nd Wirnd, F. (989). Lctte trnsport in isolted mouse muscle studied with trcer technique kinetics, stereospecificity, ph dependency nd mximl cpcity. Act Physiol. Scnd. 37, Kinsey, S. T. nd Ellington, W. R. (996). H- nd 3 P-nucler mgnetic resonnce studies of L-lctte trnsport in isolted muscle fiers from the spiny loster Pnulirus rgus. J. Exp. Biol. 99, Leree, K. nd Millign, C. L. (999). Lctte trnsport cross srcolemml vesicles isolted from rinow trout white muscle. J. Exp. Biol. 22, McCullgh, K. J. A., Poole, R. C., Hlestrp, A. P., O Brien, M. nd Bonen, A. (996). Role of the lctte trnsporter (MCT) in skeletl muscles. Am. J. Physiol. 27, E43 E5. McCullgh, K. J. A., Poole, R. C., Hlestrp, A. P., Tipton, K. F., O Brien, M. nd Bonen A. (997). Chronic electricl stimultion increses MCT nd lctte uptke in red nd white skeletl muscle. Am. J. Physiol. 273, E239 E246. McDermott, J. C. nd Bonen, A. (994). Lctte trnsport in rt srcolemml vesicles nd intct skeletl muscle nd fter muscle contrction. Act Physiol. Scnd. 5, Moerlnd, T. S. nd Kushmerick, M. J. (994). Contrctile economy nd eroic recovery metolism in skeletl muscle dpted to cretine depletion. Am. J. Physiol. 267, C27 C37. Nikolovski, S., Fulkner, D. L., Plmer, T. N. nd Fournier, P. A. (996). Muscle glycogen repletion from endogenous cron sources during recovery from high intensity exercise in the fsted rt. Act Physiol. Scnd. 57, Pilegrd, H., Terzis, G., Hlestrp, A. nd Juel, C. (999). Distriution of the lctte/h + trnsporter isoforms MCT nd MCT4 in humn skeletl muscle. Am. J. Physiol. 276, E843 E848. Poole, R. C. nd Hlestrp, A. P. (993). Trnsport of lctte nd other monocroxyltes cross mmmlin plsm memrnes. Am. J. Physiol. 264, C76 C782. Putnm, R. W., Gleeson, T. T. nd Bennett, A. F. (98). Histochemicl determintion of the fier composition of locomotory muscles in lizrd, Dipsosurus dorslis. J. Exp. Biol. 24, Roth, D. A. (99). The srcolemml lctte trnsporter: trnsmemrne determinnts of lctte flux. Med. Sci. Sports Exerc. 23, Roth, D. A. nd Brooks, G. A. (99). Lctte trnsport is medited y memrne-ound crrier in rt skeletl muscle srcolemml vesicles. Arch. Biochem. Biophys. 279, Roth, D. A. nd Brooks, G. A. (99). Lctte nd pyruvte trnsport is dominted e ph grdient-sensitive crrier in rt skeletl muscle srcolemml vesicles. Arch. Biochem. Biophys. 279, Wng, Y., Wright, P. M., Heigenhuser, G. J. F. nd Wood, C. M. (997). Lctte trnsport y rinow trout white muscle: kinetic chrcteristics nd sensitivity to inhiitors. Am. J. Physiol. 272, R577 R587. Wtt, P. W., McLennn, P. A., Hundl, H. S., Kuret, C. M. nd Rennie, M. J. (988). L(+)-Lctte trnsport in perfused rt skeletl muscle: kinetic chrcteristics nd sensitivity to ph nd trnsport inhiitors. Biochim. Biophys. Act 994, Wilson, M. C., Jckson, V. N., Heddle, C., Price, N. T., Pilegrd, H., Juel, C., Bonen, A., Montgomery, I., Hutter, O. F. nd Hlestrp, A. P. (998). Lctic cid efflux from white skeletl muscle is ctlyzed y the monocroxylte trnsporter isoform MCT3. J. Biol. Chem. 273, Wyss, M. nd Kddurh-Douk, R. (2). Cretine nd cretinine metolism. Physiol. Rev. 8, 7 23.