The role of mineralized tissue in the buffering of lactic acid during anoxia and exercise in the leopard frog Rana pipiens

Transcription

1 The Journl of Experimentl Biology 28, Pulished y The Compny of Biologists 25 doi:1.1242/je The role of minerlized tissue in the uffering of lctic cid during noxi nd exercise in the leoprd frog Rn pipiens Dniel E. Wrren* nd Donld C. Jckson Brown University, Deprtment of Moleculr Phrmcology, Physiology nd Biotechnology, Box G, Providence, RI 2912, USA *Author for correspondence (e-mil: Dniel_E_Wrren@rown.edu) Accepted 1 Jnury 25 To evlute the role of minerlized tissues of the leoprd frog in uffering cid, we nlyzed the composition of femur nd uditory cpsule, the ltter of which encloses portion of the endolymphtic lime scs, nd investigted the extent to which these tissues re involved in uffering lctic cid fter 2.5 h of noxi nd 1 19 min of strenuous exercise t 15 C. We nlyzed the following tissues for lctte: plsm, hert, liver, gstrocnemius muscle, femur, uditory cpsule nd crcss. Plsm [C 2+ ], [Mg 2+ ], [inorgnic phosphte (P i )], [N + ] nd [K + ] were lso mesured. Femur C 2+, P i nd 2 CO 3 compositions were similr to one in other vertertes. Auditory cpsule hd significntly more CCO 3 thn femur. Lctte ws significntly elevted in ll tissues fter noxi nd exercise, including femur nd Summry uditory cpsule. Anoxi incresed plsm [C 2+ ], [Mg 2+ ], [P i ] nd [K + ] nd hd no effect on plsm [N + ]. Exercise incresed plsm [Mg 2+ ], [P i ] nd [K + ] nd hd no effect on plsm [C 2+ ] or [N + ]. The skeleton nd endolymphtic lime scs uffered 21% of the totl lctte lod during noxi, nd 9% fter exercise. The exct contriution of the entire endolymphtic sc system to lctte uffering could not e determined in the present study. We conclude tht the minerlized tissues function s uffers during noxi nd exercised induced lctic cidosis in mphiins. Key words: noxi, one, uffering, C 2+, exercise, lctic cid, leoprd frog, Rn pipiens. Introduction Exhustive exercise nd environmentl hypoxi or noxi led to metolic cidosis in mphiins, chrcterized y cidemi, decresed plsm icronte nd incresed lood nd tissue lctte concentrtions (Boutilier et l., 1997; McDonld et l., 198; Toews nd Boutilier, 1986; Wegener nd Kruse, 1993). Trditionlly, in these nd most other vertertes, plsm HCO 3 is thought to e the principl extrcellulr uffer during metolic cidosis. More recently, minerlized tissue hs een shown to e importnt in uffering lctic cid during noxic sumergence in reptiles, specificlly the skeleton nd shell in pinted turtles (Jckson, 2) nd skeleton nd osteoderms in cimn (Jckson et l., 23). Reptile one uffers lctic cid in severl wys. It functions s lctte sink ecuse it hs low endogenous lctic cid production nd, therefore, lctic cid produced y other tissues nd distriuted to the extrcellulr fluid cn ccumulte in the one. When lctte ccumultes to concentrtions greter thn clculted from the tissue wter, the one is sid to sequester lctte, which is most likely y complexing to clcium in the one. Verterte one lso contins significnt quntities of clcium cronte, which when lierted, chemiclly uffer protons generted y glycolysis. The resultnt cron dioxide from this chemicl rection diffuses into the surrounding wter while the remining clcium ccumultes in the extrcellulr fluid nd cn rech high concentrtions, especilly t 3 C fter 3 months of noxic sumergence in pinted turtles (~1 mequiv l 1 ; Jckson, 22). Anurn mphiins lso possess significnt clcium cronte deposits in their endolymphtic system (Whiteside, 1922). Although restricted to the inner er in most vertertes, this system is lrge nd extends down the length of the verterl column in nurn mphiins. The exct function of the lrge endolymphtic system in frogs is not known. Proposed hypotheses include the protection of the spinl gngli, s n endolymph reservoir when pressures in the uditory lyrinth re high, or s n id in sound trnsmission (Simkiss, 1967). After he hd oserved tht C 2+ excretion incresed during environmentl hypercpni, Simkiss (1968) proposed tht the frog endolymphtic system helps mintin cid se homeostsis y relesing CCO 3. This hypothesis ws further supported in susequent study (Tufts nd Toews, 1985) in which hypercpni incresed plsm C 2+ concentrtions in tods. These investigtors estimted tht hlf of the oserved compenstory HCO 3 response must hve

2 1118 D. E. Wrren nd D. C. Jckson come from internl CCO 3 stores ecuse the levels could not e ccounted for solely y uptke cross the skin. Our purpose ws to determine whether the skeleton nd endolymphtic system of the leoprd frog Rn pipiens ply roles in uffering lctic cid ccumulted during noxic sumergence nd strenuous exercise. There re no previous reports of lctte ccumultion in minerlized tissue s result of noxi in n mphiin, or following exhustive exercise in ny verterte. Although the leoprd frog is not likely to experience noxi in its nturl environment, it is very likely to ccumulte extreme levels of lctic cid due to lesser degrees of oxygen lck during winter hierntion (Donohoe nd Boutilier, 1999). Therefore, to study the frogs response to noxi is useful for determining role for minerlized tissue responses in uffering lctic cid, which we ssessed y mesuring lctte ccumultion in the minerlized tissues nd compring it to the lctte concentrtions in other tissues of the frog. We ssessed the skeletl contriution s sources of chemicl uffering y mesuring the concentrtions of their mjor ionic constituents in plsm. Becuse it ws esy to dissect rpidly, we smpled the entire frog uditory cpsule, prt of the skull tht encloses the memrnous lyrinth nd portion of the endolymphtic sc (Fig. 1; Whiteside, 1922). The minerl compositions of femur nd uditory cpsule were nlyzed for comprisons with one of other vertertes. We lso incuted frog femur in 3 mmol l 1 lctte solutions t ph 8. nd 7.3 for 6 nd 24 h to scertin the cpcity for lctte uptke in frog one. Mterils nd methods Experimentl nimls Leoprd frogs Rn pipiens Schreer 1782 (ody mss g), were otined from Lemerger (Oshkosh, WI, USA). Frogs used in the noxi, exercise nd minerl composition experiments were held t 15 C for 3 5 dys until used in experiments. Frogs whose femurs were used in the one incution experiments were held t 6 C for 2 3 weeks until killed. The nimls were held in Providence tpwter (C 2+ =17.3 p.p.m., Mg 2+ =.6 p.p.m., N + =1. p.p.m., K + =.8 p.p.m.) nd were not fed during the course of the experiments. All prts of this experiment were pproved y the Brown University Institutionl Animl Cre nd Use Committee (IACUC) Determintion of one nd uditory cpsule C 2+, Mg 2+, N +, K + nd P i concentrtions The ionic compositions of frog one nd uditory cpsule were determined. Frogs were pithed nd their femurs (N=1 5 nimls) dissected from skeletl muscle. Auditory cpsules (N=2 5 nimls), formed y the prootic nd exoccipitl ones of the skull, were smpled y isecting the skull mid-sgitlly nd dissecting them from surrounding muscle nd the squmosl one on the lterl edge of the skull. Femur nd uditory cpsules were dried for 2 3 dys t 87 C nd ground to powder under liquid nitrogen (Spex 67, Freezer Mill, Eye Spinl cord Brin Auditory cpsule Endolymphtic system Fig. 1. Dorsl view of frog depicting the loction of the uditory cpsule. The endolymphtic system (region shded lck) is omitted from the left side. (Modified from Whiteside, 1922.) Metuchen, NJ, USA). The powder ws plced in porcelin vils nd heted to 475 C for 2 dys to urn wy ll orgnic mteril. The resultnt sh ws dissolved in 12 vol. 2 mol l 1 HCl nd nlyzed for N + nd K + using flme photometry (IL model 943; Lexington, MA, USA). The HCl ws further diluted with 5 vol. of deionized wter (6 vol. totl) nd nlyzed for Mg 2+. A portion of this solution ws further diluted with 3 vol. of deionized wter (18 vol. totl) nd nlyzed for C 2+ nd inorgnic phosphte (P i ; see elow for the detils of the nlysis). Atomic sorption spectrophotometry (Perkin-Elmer model 28, Boston, MA, USA) ws used to mesure C 2+ nd Mg 2+ levels nd stndrd kit for P i (Kit 67 Sigm, St Louis, MO, USA). 2 Determintion of one nd uditory cpsule CO 3 A known mount (.2.1 g) of one or uditory cpsule powder (otined s descried ove) ws introduced into flsk contining 15 ml 2 mol l 1 HCl through which humidified nitrogen gs ws pssed (~24 ml min 1 ; Jckson et l., 1999). Cron dioxide, generted s the result of CO 2 3 titrtion, ws then crried y the nitrogen gs through drying column (Drierite, Xeni, OH, USA) nd then through CO 2 nlyzer (AEI, model CD-3A, Pittsurgh, PA, USA). The output from the cron dioxide nlyzer ws recorded on lptop computer using dt cquisition system (BIOPAC MP1, Golet, CA, USA) nd nlyzed using softwre (Acqknowledge, BIOPAC, Golet, CA, USA). The volume of CO 2 generted ws clculted y the following: Vol. CO 2 generted = (Averge %CO 2 Time verged N 2 flow rte) / 1 All volumes were corrected to STPD nd converted to mmoles CO 2, ssuming the constnt ml per mmole CO 2 (Cmeron, 1989). One mole of CO 2 ws ssumed to e derived from one mole of CO 2 3. Anoxi nd exercise experimentl protocol The noxi nd exercise experiments were crried out t

3 Bone s uffer in leoprd frogs C. Frogs were rndomly chosen s controls, noxic or exercised. Control frogs (N=6) were plced individully into drkened, 1-liter continers (dimeter=11 cm, height=12 cm) contining.5 l erted wter. The next dy, 5 ml of uffered.1 g ml 1 MS222 solution (uffered to ph 7. with 1 mol l 1 NOH) were dded to one of the continers. After 2 min, the nesthetized niml ws removed, pithed, weighed nd smpled s descried elow. The remining frogs were similrly smpled in turn. Control frogs were treted in this mnner ecuse preliminry experiments in which we pithed frogs without nesthesi showed significnt lctte ccumultion in their tissues nd it ws our intention to smple the tissues with tissue lctte contents s low s possile. Anoxic frogs (N=6) were plced in wter-filled 15 cm 21 cm 43 cm (W H L) qurium vigorously uled with nitrogen gs for t lest 1 h prior, in order to displce ny dissolved oxygen. A plstic mesh screen ws plced just elow the surfce of the wter to prevent ccess to ir. An dditionl crylic lid covered ll ut smll prt of the wter s surfce to llow for nitrogen gs to escpe. Vigorous uling of the nitrogen gs ws continued throughout the sumergence. After 2.5 h, the nimls were removed, pithed, weighed nd smpled s descried elow. Anoxic frogs were not nesthetized ecuse the nimls were quiescent, ut still live, y the end of noxic sumergence nd did not struggle during the pithing. The quiescence hs een oserved in previous studies of noxi in frogs (Wegener nd Kruse, 1993). Exercised frogs (N=6) were plced in 15 cm 21 cm 43 cm (W H L) qurium with 2 3 cm deep wter nd chsed with metl rod until they were incple of further urst swimming (1 19 min). The nimls were removed, pithed, weighed nd smpled s descried elow. Exercised frogs were not nesthetized ecuse doing so would hve required n dditionl 2 min fter the exercise period ended nd would hve prevented us from exmining the immedite post-exercise condition of the niml. The nimls struggled little, if t ll, during the pithing ecuse they were exhusted from the exercise nd so dditionl lctte production fter the exercise period ws unlikely. Tissue smpling All smpling ws performed in cold-room t 3 C to minimize chnges in tissue metolites during dissection. After pithing, mid-line incision on the ventrl side of the frog ws mde nd lood (.1.4 ml) ws smpled vi crdic puncture nd plced on ice. Hert, liver nd gstrocnemius muscle were quickly smpled nd flsh-frozen in clmps cooled using liquid nitrogen. The femur (1 per niml) ws clered of skeletl muscle, its shft cut lengthwise nd the mrrow removed. The one frgments were quickly freeze clmped. Auditory cpsules (2 per niml) were smpled y completely isecting the skull mid-sgitlly nd cutting them free of surrounding muscle nd one (oritl nd mxill), nd were freeze clmped. The remining crcss ws clmped nd frozen y immersion in liquid nitrogen. The lood smples were kept on ice for 1 2 h until they could e centrifuged for 3 min t 93 g. Previous experience in our lortory hs shown tht the lctte nd ionic composition of the plsm do not chnge when treted in this mnner. The plsm ws trnsferred to nother vil nd stored t 25 C until nlyzed for lctte (Kit 735-1; Trinity Biotech, St Louis, MO, USA), C 2+ nd Mg 2+ (tomic sorption spectrophotometry; Perkin-Elmer model 28), N + nd K + (flme photometry; IL model 946, Lexington, MA, USA) nd P i (Kit 67, Sigm). The frozen tissues were kept on dry ice for 1 2 h efore they could e trnsferred to deep freeze, where they were stored t 75 C until nlyzed for lctte. Lctte nlyses of tissues nd plsm Frozen hert, liver nd gstrocnemius muscle (~2 mg) were homogenized in 1 ml ice-cold.6 mol l 1 perchloric cid using Mini-Bedeter 311BX (Biospec, Brtlesville, OK, USA) using 1 mm glss eds for 3 min. The frozen crcss ws homogenized in 4 vol. of ice-cold.6 mol l 1 perchloric cid using Virtis Super 3 homogenizer (Grdiner, NY, USA). Frozen femur nd uditory cpsules were ground to powder under liquid nitrogen (Spex 67, Freezer Mill). The powder ws incuted in 5 vol. of ice-cold.6 mol l 1 perchloric cid for 2 h on ice, vortexing every 2 min. Smples of ll tissue homogentes were centrifuged t 93 g for 3 min. [Lctte] ws mesured in the resultnt superntnts nd plsm using n enzymtic ssy (Kit 735-1, Trinity Biotech). Bone incutions Twelve leoprd frogs were euthnized with intrperitonel injections of Beuthnsi -D Specil (Schering-Plough, Millsorough, DE, USA) nd their femurs removed, clened of soft tissue including mrrow, nd frozen t 25 C until used in the incutions. One femur from ech of the 12 nimls ws incuted in eker contining solution of.8% NCl, 1 mmol l 1 TES nd 3 mmol l 1 lctte t ph 8., nd the other femurs in n identicl solution t ph 7.3. The volume of solution in ech eker ws 8 ml. Six femurs were smpled from ech eker fter 6 h nd 24 h, frozen in liquid nitrogen nd stored t 25 C until nlyzed for lctte s descried ove. Sttisticl nlyses Differences in the composition of femur nd uditory cpsule were determined using t-tests. Two-wy multivrite nlysis of vrince (MANOVA) ws used to determine whether tissue type nd tretment ffected lctte concentrtion. Lest-squres men (LSM) tests were used to determine whether tretment ffected the plsm concentrtions of C 2+, Mg 2+, N +, K + nd P i, nd whether time nd ph ffected lctte uptke into one in the incution experiment. Student s t-tests were used to elucidte differences reveled y the MANOVA nd LSM tests. All sttisticl nlyses were performed using JMP 4. (SAS Institute, Cry, NC, USA).

4 112 D. E. Wrren nd D. C. Jckson Tle 1. Composition of femur nd uditory cpsule of the leoprd frog Rn pipiens CO 3 2 Composition (mmol kg 1 sh) Tissue Wter (%) Orgnic (%) Ash (%) (mmol kg 1 dry tissue) P i C 2+ Mg 2+ N + K + Femur 54.3±.1* 25.5±.1* 2.2±.1* 533±24* 6192±44* 99±99* 288±4* 483± ±4.1* Auditory cpsule 7.± ± ± ± ± ±15 256±6 453± ±6.3 Vlues re mens ± S.E.M. (N=5 for ech tissue). *Significnt difference etween the two tissues (P<.5, t-test). Results Femur nd uditory cpsule composition The compositions of femur nd uditory cpsule re summrized in Tle 1. All of the inorgnic components, except for CO 3 2, re expressed s mmol kg 1 sh. CO 3 2 ws mesured on dry one powder nd is expressed s mmol kg 1 dry tissue. Auditory cpsule contined more wter nd less orgnic mtter thn femur. Femur sh contined slightly more P i nd more Mg 2+ thn uditory cpsule. There ws no difference in the mount of N + etween the two tissues. Auditory cpsule sh contined more C 2+ nd K + thn femur sh. Dry uditory cpsule contined lmost twice the CO 3 2 of dry femur. Lctte distriution in normoxic controls Tissue lctte concentrtions from nesthetized, ut otherwise undistured, nimls re shown in Fig. 2 nd were low in ll tissues exmined (mens rnged mmol kg 1 ). Lctte distriution during noxi Anoxic sumergence t 15 C for 2.5 h significntly incresed lctte concentrtions ove control levels in ll tissues, including femur nd uditory cpsule (Fig. 2). Femur nd uditory cpsule lctte concentrtions (15. nd 13.2 mmol kg 1, respectively) were similr to those in crcss (13.5 mmol kg 1 ). Gstrocnemius muscle ccumulted the most lctte (24.7 mmol kg 1 ) nd liver the lest (9. mmol kg 1 ), oth of which were significntly different from the other tissues. Plsm nd hert ccumulted similr lctte concentrtions (2.4 nd 18.4 mmol kg 1, respectively), which were significntly greter thn ll other tissues except gstrocnemius muscle. Lctte distriution during exhustive exercise The men time to exhustion ws 13.1±1.6 min (rnge=1 19 min) t 15 C. Lctte concentrtions were significntly elevted reltive to controls in ll tissues, including femur nd uditory cpsule (Fig. 2). Femur lctte concentrtions were similr to those in liver nd uditory cpsule nd were significntly lower thn the other tissues. Auditory cpsule lctte concentrtions were significntly less thn in plsm, gstrocnemius nd hert, ut similr to ll other tissues. Gstrocnemius muscle nd plsm ccumulted the most lctte (13.9 nd 14. mmol kg 1, respectively) nd femur nd liver the lest (5.3 nd 4.6 mmol kg 1, respectively). The lctte concentrtion in plsm did not differ from tht in hert (1.6 mmol kg 1 ). Plsm ion chnges during noxi nd exhustive exercise Plsm ion concentrtions during noxi nd exhustive exercise re summrized in Figs 3 nd 4. Plsm [C 2+ ] incresed significntly reltive to controls fter 2.5 h noxi, ut ws unffected y exhustive exercise. Plsm [Mg 2+ ] incresed in oth noxic nd exercise groups y 35% nd 17%, respectively, nd ws significntly different cross ll groups. Plsm [P i ] nd [K + ] incresed significntly in oth noxic nd exercised frogs ut did not differ etween the two groups. Plsm [N + ] ws unffected y noxi or exercise. [Lctte] (mmol kg 1 ) Control 2 h noxi Exercised Plsm Crcss Aud. Cps.,c c d c c Femur Gstroc. Liver Hert Bone incutions The results of the one incutions re presented in Fig. 5 nd show tht frog one sequestered lctte t ph 8. nd 7.3. There ws no significnt interction etween ph nd time Fig. 2. Lctte concentrtion in plsm, crcss, uditory cpsule (Aud. Cps.), femur, gstrocnemius muscle (Gstroc.), liver nd hert in controls, fter 2 h noxi nd fter exhustive exercise in leoprd frogs t 15 C. Lctte concentrtions fter noxi nd exercise re significntly higher thn controls for ll tissues. Vlues re mens ± S.E.M., N=5 6 per tretment for ech tissue. Differences etween tissues within tretment re indicted y differing letters (two-wy MANOVA, Student s post-hoc t-test)

5 Bone s uffer in leoprd frogs 1121 Plsm [C 2+ ] (mmol l 1 ) A Plsm [K + ] (mmol l 1 ) A Plsm [Mg 2+ ] (mmol l 1 ) Plsm [P i ] (mmol l 1 ) B C (P=.7), ut time nd ph, y themselves, hd significnt effects. Lctte concentrtions of the solutions fell during incution from 29.7 to 27.3 mmol l 1 t ph 7.3 nd from 29.3 to 27.9 mmol l 1 t ph 8.. Discussion This study extends previous oservtions on lctte uptke into one during noxi in other nimls (Jckson, 2; Jckson et l., 23, 21), ut is the first report of uptke during noxi in n mphiin nd following out of exhustive exercise in ny verterte. We hve demonstrted tht the mphiin skeletl nd endolymphtic systems ccumulte lctte during oth noxic sumergence nd exhustive exercise nd cn do so in s little s 1 min, the Control 2 h noxi Exercise Fig. 3. Plsm concentrtions of (A) C 2+, (B) Mg 2+ nd (C) P i in controls, fter 2 h noxi nd fter exhustive exercise in leoprd frogs t 15 C. Vlues re mens ± S.E.M., N=6 per tretment for ech ion. Differences etween tretments re indicted y differing letters (LSM, Student s post-hoc t-test). c Plsm [N + ] (mmol l 1 ) B Control 2 h noxi Exercise Fig. 4. Plsm concentrtions of (A) K + nd (B) N + in controls, fter 2 h noxi nd fter exhustive exercise in leoprd frogs t 15 C. Vlues re mens ± S.E.M., N=5 per tretment for ech ion. Differences etween tretments re indicted y differing letters (LSM, Student s post-hoc t-test). fstest time oserved in ny verterte exmined. Bsed on chnges in plsm [C 2+ ], the skeleton nd/or endolymphtic systems lso functioned s sources of chemicl uffer for the extrcellulr fluid during noxic sumergence, ut not exercise, in our study. Our dt further demonstrte tht uffering of lctic cid is generl property of minerlized tissue. The composition of frog one in this study is similr to tht in previous nlysis nd to most other vertertes (Biltz nd Pellegrino, 1969). Frog femur contins less CO 3 2- thn one nd shell of the extremely noxi-tolernt pinted turtle Chrysemys pict ellii (Jckson et l., 2), nd osteoderms of the rod-nosed cimn (Jckson et l., 23). Auditory cpsule hs CCO 3 composition tht is similr to tht in the long one nd shell of the pinted turtle Chrysemys pict ellii (Jckson et l., 2) nd greter thn cimn osteoderm (Jckson et l., 23). The lrge CCO 3 deposits in our nlysis of uditory cpsule re lso consistent with the erliest descriptions of the endolymphtic lime deposits (Dempster, 193; Whiteside, 1922) nd indicte tht we succeeded in smpling them with our protocol. This verifiction is importnt ecuse (1) our smpling included the derml one encpsulting portion of the endolymphtic lime sc, nd (2) we used the sme smpling technique in our lctte nlyses fter noxi nd exercise. The

6 1122 D. E. Wrren nd D. C. Jckson Femur lctte concentrtion (mmol kg 1 wet one) ph 7.3 ph Incution time (h) Fig. 5. Concentrtion of lctte (mmol kg 1 wet mss) in frog femurs incuted in 3 mmol l 1 lctte for 6 nd 24 h t ph 8. or 7.3. Vlues re mens ± S.E.M., N =6 per tretment t ech time point. There ws no significnt time ph interction ut there were seprte ph nd time effects (LSM, Student s post-hoc t-test). smpled structure hd milky ppernce tht ws consistent with erlier descriptions of the endolymphtic system. Both exercise nd noxi incresed lctte concentrtions in ll tissues exmined to levels oserved in previous studies (Andersen nd Wng, 23; Armentrout nd Rose, 1971; Bennett nd Licht, 1974; D Eon et l., 1978; Donohoe nd Boutilier, 1999; Hutchison nd Turney, 1975; Wrurton et l., 1989; Wsser et l., 1993, 1991; Wegener nd Kruse, 1993). With the exception of gstrocnemius nd hert, plsm hd higher lctte concentrtion thn the other tissues exmined, s hs lso een oserved in cold hypoxic frogs (Donohoe nd Boutilier, 1999) nd cold noxic turtles (Jckson et l., 1996). High lctte levels in gstrocnemius nd hert cn e ttriuted to their intense ctivity during the exercise nd during the initil period in the noxi chmer. The frogs ecme quiescent out 3 min into the 2 h noxi out. Higher extrcellulr thn intrcellulr lctte concentrtions hve een reported during cold hypoxi in frogs y Donohoe nd Boutilier (1999), who suggested tht lctte is trnsported from loclly neroic tissues, where it is produced, to loclly eroic tissues, where it cn e oxidized or converted to glucose. In the noxic frogs with no eroic tissues, we propose tht exporting lctte from the cells to the extrcellulr fluid (ECF) my etter exploit the uffer cpcity of the minerlized tissues, the skeleton nd endolymphtic system. Three pieces of evidence reveled y the present study suggest potentilly importnt role for the skeleton in uffering lctic cid fter exercise nd noxi. First, the skeleton nd uditory cpsule ccumulted significnt mounts of lctte fter oth exercise nd noxi. The post-exercise lctte ccumultion in frog one is notle ecuse it is the first of its kind to e reported nd it occurred in s few s 1 min, rte not seen efore in vivo in ny verterte. In the cse of noxi, the minerlized tissues ccumulted more lctte thn the liver nd the lctte concentrtion in femur (15. mmol l 1 ) ws greter thn wht could e ccounted for sed on its wter composition (54%) nd the plsm lctte concentrtion (2.4 mmol l 1 ), indicting tht lctte ws chemiclly ound to minerlized one, most likely complexed with C 2+, s previously suggested to occur in pinted turtle shell (Jckson, 2). Although the post-exercise femurs nd uditory cpsules nd the noxic uditory cpsules did not ccumulte more lctte thn predicted from their wter contents, sequestrtion cnnot e ruled out. This is especilly true in the post-exercise femurs, which ccumulted exctly wht might e predicted from the wter content nd plsm [lctte]. The prediction is likely to e n overestimte ecuse it ssumes tht ll wter is extrcellulr nd in equilirium with plsm. Second, our in vitro incution of frog femur demonstrtes tht significnt sequestrtion of lctte is possile. These results revel the ccumultion tht is possile t equilirium, lthough the in vivo kinetics of exchnge in these experiments did not permit full equilirtion to occur. The in vitro one lctte levels reched in the frog re similr to mesurements on pinted turtle (D.C.J., unpulished oservtion) nd greter thn pulished uptkes in cimn osteoderms (Jckson et l., 23) nd cryfish crpce (Jckson et l., 21). The third piece of evidence tht frog one contriutes to the uffering of lctic cid is tht plsm [C 2+ ] ws elevted fter noxic sumergence. We interpret this s the result of CCO 3 relese from minerlized tissues. It is unlikely tht the chnges in plsm [C 2+ ] were cused y hemoconcentrtion ecuse plsm [N + ] did not chnge. It is notle tht n erlier study of noxi in ullfrogs did not oserve n increse in plsm [C 2+ ] (Wrurton et l., 1989). However, the frogs in tht study were prlyzed with succinylcholine, nd lctte levels were only hlf of wht we oserved, suggesting tht the severity of the cidosis my hve een insufficient to deminerlize one. The increse in plsm [P i ], during oth noxi nd exercise is most likely derived from ctive muscle s result of cretine phosphte hydrolysis during urst swimming nd noxic sumergence (Wegener nd Kruse, 1993), rther thn from one. In vitro studies show tht CO 3 2 nd not P i is the principle uffer nion relesed from oth mouse clvrie (Bushinsky et l., 22) nd turtle shell powder (Jckson et l., 1999) when incuted in cid solutions. The quntittive importnce of the frog s skeleton in uffering lctic cidosis cn e estimted y summing the frctions of the totl lctte lod tht ccumulted in the skeleton nd tht ws uffered y CCO 3 relesed from the skeleton nd/or endolymphtic system. The sis for this clcultion is the ssumption tht ech mole of lctte ccumulted in frog one is ccompnied y proton, s is the cse in turtle one (Jckson et l., 1999). We ssume tht for ech mole of C 2+ relesed from the skeleton, mole of CO 3 2, which uffers 2 moles of H + derived from glycolyis, is lso relesed. If frog s wet skeletl mss is 16% of totl ody mss (D. E. Wrren nd D. C. Jckson, unpulished), then the percentge of the totl lctte lod contined within the

7 Bone s uffer in leoprd frogs 1123 skeleton t the end of noxi nd exercise is 18% nd 9%, respectively. Unfortuntely, similr clcultion cnnot e mde for the endolymphtic system ecuse we did not smple it entirely, lthough we ssume it to e much smller thn the skeleton. If we ssume tht ll the relesed C 2+ distriutes throughout the extrcellulr fluid nd tht the extrcellulr fluid volume is 26% of ody mss (Thorson, 1964), then 3% of the totl lctte lod ws uffered y CO 3 2 relesed from the skeletl or endolymphtic systems during noxi. There is no evidence tht the skeletl nd endolymphtic systems relese chemicl uffers fter exercise ecuse plsm [C 2+ ] ws not elevted. After summtion, we estimte tht 21% nd 9% of the totl lctte lods produced during noxi nd exercise, respectively, re uffered y minerlized tissue. These estimtes hve severl importnt implictions tht require more investigtion. Although the quntittive contriution to lctic cid uffering is modest in exercise, the rpidity with which these minerlized tissues were recruited hs not een oserved previously. In ddition, the mount nd integrity of frog s minerlized tissues my determine the severity of lctic cidosis tht n niml cn tolerte. This might help to explin why frogs, lthough considered hypoxi tolernt, re noxi intolernt. Under hypoxic conditions, lctic cid is produced in loclly neroic tissue nd moves into the extrcellulr fluid where it distriutes to loclly eroic tissues to e oxidized, resulting in slower lctte ccumultion nd glycogen depletion rtes (Donohoe nd Boutilier, 1999). Although the extrcellulr distriution of lctte enles exploittion of the skeletl nd endolymphtic uffers during noxi, only modest frction of the lctte lod is uffered, primrily ecuse of the structures smll size reltive to ody mss. Therefore, only the lower rtes of lctte ccumultion tht occur during hypoxi re sustinle while the higher rtes during noxi overwhelm the limited uffering cpcity of the frog. One intriguing conservtion impliction is tht if minerlized tissues proved vulnerle to deminerliztion in n cidic environment, then the tolernce of frogs to noxi or hypoxi nd, possily, neroic performnce, could e compromised. Indeed, cidifiction of frog environments hs een implicted s cuse of mphiin decline nd low environmentl ph hs een shown to increse mortlity rtes in this species (Brodkin et l., 23; Simon et l., 22). In conclusion, the leoprd frog skeletl nd endolymphtic systems contriute uffering during exercise y rpidly functioning s sink for smll mount of the lctte lod. Their uffering roles re more importnt during noxi when these structures, especilly one, sequester lrger frction of the lctte lod s well s relese chemicl uffers. Future studies should investigte whether these systems re significnt contriutors to lctic cid uffering under the more ecologiclly relevnt conditions of long-term hypoxi t cold tempertures, nd determine how ph ffects one nd lime sc deminerliztion in mphiins. Additionl work should exmine the reltive contriutions of skeleton nd endolymphtic lime deposits to overll chemicl uffer relese. A etter ssessment of the size of the endolymphtic lime scs is needed to determine their exct contriution to lctic cid uffering. This study ws supported y NSF grnt IBN (D.C.J.). References Andersen, J. B. nd Wng, T. (23). Crdiorespirtory effects of forced ctivity nd digestion in tods. Physiol. Biochem. Zool. 76, Armentrout, D. nd Rose, F. L. (1971). Some physiologicl responses to noxi in the gret plins tod, Bufo cogntus. Comp. Biochem. Physiol. 39A, Bennett, A. F. nd Licht, P. (1974). Aneroic metolism during ctivity in mphiins. Comp. Biochem. Physiol. 48A, Biltz, R. M. nd Pellegrino, E. D. (1969). The chemicl ntomy of one. I. A comprtive study of one composition in sixteen vertertes. J. Bone Joint Surg. Am. 51, Boutilier, R. G., Donohoe, P. H., Tttersll, G. J. nd West, T. G. (1997). Hypometolic homeostsis in overwintering mphiins. J. Exp. Biol. 2, Brodkin, M., Vtnick, I., Simon, M., Hopey, H., Butler-Holston, K. nd Leonrd, M. (23). Effects of cid stress in dult Rn pipiens. J. Exp. Zool. A Comp. Exp. Biol. 298, Bushinsky, D. A., Smith, S. B., Gvrilov, K. L., Gvrilov, L. F., Li, J. W. nd Levi-Setti, R. (22). Acute cidosis-induced ltertion in one icronte nd phosphte. Am. J. Physiol. 283, F191-F197. Cmeron, J. N. (1989). The Respirtory Physiology of Animls. New York: Oxford University Press. Dempster, W. T. (193). The morphology of the mphiin endolymphtic orgn. J. Morphol. Physiol. 5, D Eon, M. E., Boutilier, R. G. nd Toews, D. P. (1978). Aneroic contriutions during progressive hypoxi in the tod Bufo mrinus. Comp. Biochem. Physiol. 6A, 7-1. Donohoe, P. H. nd Boutilier, R. G. (1999). The use of extrcellulr lctte s n oxidtive sustrte in the oxygen-limited frog. Resp. Physiol. 116, Hutchison, V. H. nd Turney, L. D. (1975). Glucose nd lctte concentrtions during ctivity in the leoprd frog, Rn pipiens. J. Comp. Physiol. 99, Jckson, D. C. (2). How turtle s shell helps it survive prolonged noxic cidosis. News Physiol. Sci. 15, Jckson, D. C. (22). Hiernting without oxygen: physiologicl dpttions of the pinted turtle. J. Physiol. (Lond.) 543, Jckson, D. C., Andrde, D. V. nd Ae, A. S. (23). Lctte sequestrtion y osteoderms of the rod-nose cimn, Cimn ltirostris, following cpture nd forced sumergence. J. Exp. Biol. 26, Jckson, D. C., Crocker, C. E. nd Ultsch, G. R. (2). Bone nd shell contriution to lctic cid uffering of sumerged turtles Chrysemys pict ellii t 3 C. Am. J. Physiol. 278, R1564-R1571. Jckson, D. C., Golderger, Z., Visuri, S. nd Armstrong, R. N. (1999). Ionic exchnges of turtle shell in vitro nd their relevnce to shell function in the noxic turtle. J. Exp. Biol. 22, Jckson, D. C., Toney, V. I. nd Okmoto, S. (1996). Lctte distriution nd metolism during nd fter noxi in the turtle, Chrysemys pict ellii. Am. J. Physiol. 271, R49-R416. Jckson, D. C., Wng, T., Koldkjer, P. nd Tylor, E. W. (21). Lctte sequestrtion in the crpce of the cryfish Austropotmoius pllipes during exposure in ir. J. Exp. Biol. 24, McDonld, D. G., Boutilier, R. G. nd Toews, D. P. (198). The effects of enforced ctivity on ventiltion, circultion nd lood cid-se lnce in the semi-terrestril nurn, Bufo mrinus. J. Exp. Biol. 84, Simkiss, K. (1967). Endolymphtic scs. In Clcium in Reproductive Physiology, pp New York: Rheinhold. Simkiss, K. (1968). Clcium nd cronte metolism in the frog (Rn temporri) during respirtory cidosis. Am. J. Physiol. 214, Simon, M. P., Vtnick, I., Hopey, H. A., Butler, K., Korver, C., Hilton, C., Weimnn, R. S. nd Brodkin, M. A. (22). Effects of cid exposure on nturl resistnce nd mortlity of dult Rn pipiens. J. Herpetol. 36,

8 1124 D. E. Wrren nd D. C. Jckson Thorson, T. B. (1964). The prtitioning of ody wter in Amphii. Physiol. Zool. 37, Toews, D. P. nd Boutilier, R. G. (1986). Acid-Bse Regultion in the Amphii. In Acid Bse Regultion in Animls (ed. N. Heisler), pp New York: Elsevier. Tufts, B. nd Toews, D. (1985). Prtitioning of regultory sites in Bufo mrinus during hypercpni. J. Exp. Biol. 119, Wrurton, S. J., Wsser, J. S. nd Jckson, D. C. (1989). Crdiovsculr nd metolic responses during noxic sumergence in the ullfrog with nd without mintined extrcellulr ph. J. Exp. Zool. 251, Wsser, J. S., Jckson, D. C., Chng, S. Y. nd Wrurton, S. J. (1993). Mintennce of high extrcellulr ph does not influence cell ph or metolism in sumerged noxic ullfrogs. J. Exp. Zool. 265, Wsser, J. S., Wrurton, S. J. nd Jckson, D. C. (1991). Extrcellulr nd intrcellulr cid-se effects of sumergence noxi nd nitrogen rething in turtles. Respir. Physiol. 83, Wegener, G. nd Kruse, U. (1993). Environmentl nd exercise neroiosis in frogs. In Surviving Hypoxi (ed. P. W. Hochchk, P. L. Lutz, T. Sick, M. Rosenthl nd G. vn den Thillrt), pp Boc Rton: CRC Press. Whiteside, B. (1922). The development of the sccus endolymphticus in Rn temporri Linne. Am. J. Ant. 3,