mitochondrial citrulline synthesis in the ureagenic toadfish,opsanus beta, is dependent on carbonic...

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 279:521–529 (1997)

© 1997 WILEY-LISS, INC.

JEZ 867

Mitochondrial Citrulline Synthesis in the UreagenicToadfish, Opsanus beta, Is Dependent on CarbonicAnhydrase Activity and Glutamine Transport

RAYMOND P. HENRY1* AND PATRICK J. WALSH2

1Department of Zoology and Wildlife Science, Auburn University, Auburn,Alabama 36849-5414

2Division of Marine Biology and Fisheries, Rosenstiel School of Marine andAtmospheric Science, University of Miami, Miami, Florida 33149-1098

ABSTRACT Mitochondria isolated from the liver of the Gulf toadfish Opsanus beta producescitrulline in the presence of 5 mM glutamine. Citrulline production was inversely related tosuccinate concentration between 0 and 10 mM, with a maximal rate being achieved at 0.1 mM.When toadfish are induced to become ureagenic by crowding-associated stress, mitochondrialcitrulline production increases by approximately 10-fold. Citrulline synthesis is dependent onintramitochondrial carbonic anhydrase (CA) activity, being inhibited by about 75% by both aceta-zolamide and methazolamide. The addition of exogenous CA did not increase mitochondrial cit-rulline production. Anesthetizing the fish with MS 222 prior to mitochondrial isolation resultedin the near elimination of the capacity for citrulline synthesis. Mitochondria were also shown topossess an inducible glutamine transport system. The Vmax for glutamine uptake increased three-fold and the Km increased four-fold in ureagenic vs. ammoniogenic toadfish. The transport sys-tem is the second labile component of the overall ornithine-urea cycle to be identified, and itprovides a link between the production of glutamine via cytoplasmic glutamine synthetase andits consumption via mitochondrial carbamoyl phosphate synthetase III. J. Exp. Zool. 279:521�529, 1997. © 1997 Wiley-Liss, Inc.

The Gulf toadfish, Opsanus beta, is one of thefew teleost fish that has the capacity to becomeureotelic on a facultative basis. Under conditionsof minimal stress (i.e., individual fish isolated inlarge holding tanks) toadfish are primarily am-monotelic, excreting 61–80% of their total nitro-gen as ammonia, depending on their nutritionalstatus (starved vs. fed, Walsh and Milligan, ’95).However, when animals are either crowded to-gether or confined individually in small, restrictedchambers, their pattern of nitrogen excretion isaltered such that urea becomes the predominantexcretory product, accounting for 90% of the totalnitrogen excreted (Walsh et al., ’94; Walsh andMilligan, ’95).

Unlike ammonia, which is excreted in a con-tinuous, uniform fashion, urea excretion in toad-fish occurs in a periodic manner; most of theexcreted urea is released in a single pulse of lessthan 3 h duration aproximately once every 24 h(Walsh et al., ’90; Wood et al., ’95). The pulse isreleased from the head region and is believed tooriginate from the gills (Wood et al., ’95).

While the properties of urea excretion have been

extensively characterized in this species, muchless is known about the mechanism, control, andpotential lability of urea synthesis. Toadfish areknown to possess a functional and complete he-patic ornithine-urea cycle with a glutamine-depen-dent carbamoyl phosphate synthesis (CPSase III;Mommsen and Walsh, ’89; Anderson and Walsh,’95). The transition from ammonotelism to ureo-telism involves the induction of the enzymeglutamine synthetase (GSase) in the liver whichis complete by 24 h after crowding or confinement(Walsh et al., ’94; Hopkins et al., ’95). As a result,ammonia excretion drops to near zero as ammo-nia-nitrogen is channeled into the ornithine-ureacycle as glutamine (Walsh et al., ’90; Hopkins etal., ’95; Walsh and Milligan, ’95; Anderson andWalsh, ’95). Once the transition to ureotelism iscomplete, urea synthesis appears to occur at a con-stant rate with only urea excretion being pulsa-

*Correspondence to: Raymond P. Henry, Dept. of Zoology and Wild-life Science, 101 Cary Hall, Auburn University, Auburn, AL 36849-5414. E-mail: [email protected]

522 R.P. HENRY AND P.J. WALSH

tile (Wood et al., ’97). However, it is not known ifother components of the urea pathway are alteredas well during that transition in order to achievehigher rates of synthesis. One potential point ofcontrol could occur between GSase and CPSaseIII. There are both cytoplasmic and mitochondrialisozymes of GSase (Walsh, ’97a), and it is thecytoplasmic isozyme which is induced (Milliganand Walsh, ’95). Because CPSase III is mito-chondrial, it has been speculated that one pointof control could occur via the regulation ofglutamine transport across the mitochondrialmembrane (Walsh, ’97b).

Very little is known about the intramitochondrialaspects of urea synthesis in toadfish as well, al-though it is clear that they are capable of citrullinesynthesis under some circumstances (Anderson andWalsh, ’95). Studies on isolated hepatocytes fromtoadfish have documented significant levels of ureasynthesis, showing that CPSase III is dependenton GSase for nitrogen and on carbonic anhydrase(CA) for HCO3

– (Walsh et al., ’89). Inhibition of to-tal hepatocyte CA activity by acetazolamide reducedurea synthesis by a maximum of about 70%, butsince both the cytoplasm and mitochondria containCA activity, it could not be determined if CA in oneor both subcellular compartments was necessary forHCO3

– supply to CPSase III. Some evidence frommammalian systems exists suggesting that CO2 pro-duced endogenously within the mitochondria maynot be sufficient to meet the metabolic carbon de-mand for urea synthesis. The rate of urea synthe-sis was reduced by about 60% in isolated guineapig hepatocytes when external CO2 was reducedfrom 25 mM to zero (Dodgson and Forster, ’86). Fur-thermore, Tomera et al. (’82) reported that the rateof 14C fixation from NaH14CO3 into urea in perfusedrat liver was much higher than the net rate of CO2production. This suggests that cytoplasmic CO2 maybe diffusing into the mitochondrial matrix to sup-port urea synthesis. If so, cytoplasmic CA may beimportant in maintaining the transport of CO2across the mitochondrial membrane (reviewed byGros and Dodgson, ’88; Henry, ’96).

This investigation reports on the mitochondrialcomponent of urea synthesis in the toadfish liver.Citrulline synthesis was used as an indicator ofmitochondrial nitrogen metabolism in order to elu-cidate the factors influencing the induction andregulation of urea synthesis.

MATERIALS AND METHODSCollection and maintenance of animals

Adult Gulf toadfish Opsanus beta (Goode andBean) were collected with a roller trawl in Bis-

cayne Bay, Florida, by commercial shrimpersduring April and May, 1996, for the metabolic ex-periments, and between July and October, 1996,for the transport experiments. Fish were housedin 80 L aquaria with flowing aerated seawater(detailed procedures given by Walsh and Milligan,’95). Individuals were fed shrimp but were starvedfor a period of 72 h before being used in an ex-periment.

Fish were initially held under conditions of lowpopulation density (uncrowded). High populationdensity (crowding) was used to initiate ureo-genesis: fish were transferred to 6 L plastic tubs(30 cm long x 25 cm wide × 10 cm high) with flow-ing seawater such that there were four to six in-dividuals per tub (Walsh et al., ’94). Fish werekept under these conditions for a minimum of 4days before being used.

Mitochondria preparationFish were killed by a blow to the head either

with or without having first been lightly anes-thetized in MS 222 (tricaine methanesulfonatebuffered with NaHCO3). The liver was dissectedout, and blood vessels and connective tissue werecleaned away. Fish were used individually whenlarge enough to yield 1.5 gm or more of liver; oth-erwise, livers for two to three fish were pooled.

Mitochondria were prepared according to themethod outlined by Anderson and Walsh (’95).Briefly, the liver was suspended in 5.5 volumes ofhomogenization buffer (250 mM sucrose, 0.5 mMEGTA, 5 mM K2HPO4, 30 mM HEPES, pH = 7.4at 22°C), minced with scissors, and homogenizedusing a motor-driven teflon-glass homogenizer(four to six passes). The homogenate was centri-fuged for 10 min at 750 × g (SS-34 rotor, SorvallRC5-B superspeed) at 4°C. The supernatant wassaved and centrifuged at 14,500 g for 10 min toproduce the initial mitochondrial pellet. This waswashed once in nine volumes of homogenizationbuffer and recentrifuged as above. The final pel-let was resuspended in a volume of homogeniza-tion buffer plus bovine serum albumin (BSA, 5mg ml–1), made fresh daily, equivalent to 0.3 mlg–1 of starting weight of liver.

Carbonic anhydrase activityA 50 µl sample from each preparation was

saved, frozen at –80°C, and shipped to Auburnon dry ice for analysis of CA activity. Sampleswere thawed on ice, sonicated, and measuredusing the electrometric delta pH assay (Henry,

MITOCHONDRIAL CITRULLINE SYNTHESIS IN TOADFISH 523

’91). CA activity was reported on the basis offresh weight of tissue.

Citrulline assayThe reaction medium for the analysis of mi-

tochondrial citrulline synthesis was slightlymodified from that used by Anderson and Walsh(’95; see below). This was made fresh daily andconsisted of the following: 88 mM sucrose, 0.175mM EGTA, 6 mM K2HPO4, 90 mM KCl, 5 mMNaHCO3, 2 mM MgCl2, 0.15 mM ATP, 10 mM or-nithine, 0.1 mM succinate, and 36 mM HEPES,pH = 7.40 at 22°C. Seventy five microliters of themitochondrial suspension were added to 500 µl ofthe reaction medium in a 1.5 ml microcentrifugetube; this was allowed to equilibrate to room tem-perature (22°C) for 20 min. The reaction wasstarted by adding 50 µl of a 50 mM glutamine(GLN) stock solution and gently mixing. After 30min the reaction was stopped by the addition of10 µl of 70% perchloric acid (PCA). The tubes werecentrifuged for 30 sec at 10,000 g, and the super-natant was assayed for citrulline concentration.

The citrulline assay reagent consisted of a 1:1(v:v) mixture of antipyrene (2,3-dimethyl-1-phe-nyl-3pyrazolin-5-One, 200 mg in 94 ml H2O, 6 mlconcentrated H2SO4, stored in a dark bottle atroom temperature) and diacetyl monoxime (900mg 2,3 butanedione monoxime in 95 ml H2O, 5ml glacial acetic acid, stored refrigerated in a darkbottle) made fresh before use. One ml of this re-agent was added to 50 µl of sample in a glasstube, mixed, loosely capped with a marble, andboiled in the dark for 30 min. The tubes were thencooled in an ice bath in the dark. Samples weretransferred to semi-micro cuvettes, and absor-bance was read at 464 nm (Perkin-Elmer Lambda2). The increase in citrulline concentration wasdetermined by comparison against a standardcurve of 0 to 50 µM citrulline. All chemicals werepurchased from Sigma (St. Louis, MO) and werereagent grade.

Experimental protocolCitrulline synthesis was measured for mitochon-

dria from toadfish that had been held either un-der uncrowded conditions for at least 7 days orcrowded conditions for a minimum of 4 days. Be-cause the initial measurements of the rates of cit-rulline synthesis were very low in both groups,we reinvestigated the assay conditions used pre-viously (Anderson and Walsh, ’95). Both the lin-earity of citrulline accumulation over time and therate of citrulline synthesis were tested against

varying concentrations of GLN and succinate. Fur-thermore, even after the optimal assay conditionswere established, citrulline synthesis within atreatment group of fish was at times highly vari-able, with some individual preparations showingno apparent citrulline synthesis. In order to de-termine if this was a result of the anesthetic (MS222) depressing one or more components of thecitrulline pathway, synthesis was measured onmitochondria prepared from fish that were eitherkilled after being anesthetized using MS 222 orkilled immediately without anesthesia.

For the remainder of the experiments, toadfishwere killed without prior exposure to MS 222. Mi-tochondria from these preparations were used todetermine if citrulline synthesis was dependenton mitochondrial and/or cytoplasmic CA activity.The CA inhibitors acetazolamide (Az) and meth-azolamide (Mz) were used to inhibit total CA ac-tivity (within the mitochondrial matrix and anyresidual CA activity in the general reaction me-dium). Stock solutions (10 mM) were made up inreaction medium; these were added to the finalmitochondrial suspensions (also in reaction me-dium) in a 1:10 dilution to give a working concen-tration of 1 mM. The mitochondria were allowedto incubate at room temperature for 30 min, andthe reaction was started by the addition of GLN.Another CA inhibitor, quaternary ammonium sul-fanilamide (QAS), was used to selectively inhibitCA activity in the reaction medium, leaving CAwithin the matrix functional (Henry, ’87). A stocksolutin of QAS in reaction medium (100 mM) wasdiluted 1:10 for a final working concentration of10 mM; mitochondria were incubated for 30 minbefore GLN addition.

In order to determine the potential importanceof extra-mitochondrial (i.e., cytoplasmic) CO2 incitrulline synthesis, exogenous CA activity wasadded to the reaction medium. Purified bovine redcell CA (BCA) was added to the reaction mediumto a final concentration of 0.1 mg ml–1. In a sepa-rate set of experiments, a series of HCO3

– concen-trations in the reaction medium (0, 5, and 25 mM)was used in order to determine the sensitivity ofcitrulline production to external CO2 concentra-tions. These measurements were carried out inthe presence of 0.1 mM succinate with and with-out BCA, and in the absence of succinate with andwithout BCA.

For each experiment, a blank consisting of thefinal mitochondrial suspension without the addi-tion of GLN was carried out to control for pre-existing levels of citrulline.


Mitochondrial glutamine transport

Mitochondria were isolated as above with slightmodification. Livers from four to five fish werepooled to give 3.5 to 5 g starting weight. The ho-mogenization medium consisted of 250 mM su-crose, 0.5 mM EGTA and 30 mM HEPES, pH =7.40 at 22°C. The washed mitochondrial pellet wassuspended to 0.15 ml g–1 original starting weightusing homogenization buffer plus 5 mg ml–1 BSAand kept on ice until use.

The rapid mixing/rapid filtration method ofGoldstein and Boylan (’78) was used to measurethe rate of GLN uptake. In general, 50 µl of themitochondrial suspension was drawn into a 100µl Hamilton syringe followed by 50 µl of mitochon-drial transport medium (99 mM sucrose, 0.175mM EGTA, 90 mM KCl, 2 mM MgCl2, 36 mMHEPES, pH = 7.40 at 22°C plus the metabolic in-hibitors 2 µg ml–1 rotenone and 1.5 µg ml–1 anti-mycin A). The contents were ejected into a 1.5 mlmicrocentrifuge tube and quickly withdrawn againto facilitate mixing of the mitochondria with thetransport medium. The mixture was allowed tostand for 1 min prior to injection into the uptakevessel in order to facilitate the action of the meta-bolic inhibitors. A second syringe contained 100µl of the mitochondrial transport medium plus 0.2µCi of L-[14C(U)]-GLN (200 mCi mmol–1, NEC-451,DuPont NEN) and twice the appropriate final con-centration of unlabeled GLN. The first experimen-tal series involved a fixed concentration of 0.2 mMGLN and variable uptake times between 2.5 and20 sec to determine the appropriate time to usefor kinetic measurements. The second experimen-tal series used variable final GLN concentrationsbetween 0.2 and 5 mM and a fixed uptake time of4 sec. For sucrose space determinations, 0.2 µCiof [14C(U)]-sucrose (4.5 mCi mmol–1, NEC-100, Du-Pont NEN) was substituted for GLN in the trans-port medium. Briefly, the procedure involved thevery rapid and simultaneous injection of both sy-ringes into a small mixing chamber, the bottomof which is fitted with a Millipore filter supportedon a wire mesh (see Goldstein and Boylan, ’78 fordetails of construction of the apparatus). Imme-diately after mixing, vacuum was applied to thebottom of the chamber, trapping the mitochondriaon the filter; the filter was then washed with 10ml of isolation medium containing no substrate.The entire procedure was completed within 2.5sec in our hands. The filter was then rapidly re-moved and placed in 10 ml Ecolume (ICN) andcounted in a Tracor Analytic LSC. Uptake rates

of GLN were calculated according to the equationsof Goldstein and Boylan (’78) as follows:

matrix GLN dpms = [GLNf] –[SUCROSEf/SUCROSEm] × [GLNm] (1)

where GLNf = the total GLN dpms of the filter,SUCROSEf = the dpms on the filter in the su-crose incubations, SUCROSEm = the total sucrosedpms in the medium, and GLNm = the GLN dpmsin the medium. And:

nmol GLN matrix = matrix GLNdpms/GLN specific activity (2)

where specific activity is expressed as dpmsnmol–1. Uptakes were then divided by 4 sec and nor-malized on a per mg mitochondrial protein basis.

Protein was determined on an aliquot of mito-chondrial suspension by sonicating 1:1 in 50 mMHEPES with 0.1% Triton X-100 followed by as-say according to the bicinchoninic acid method(Pierce Chemical Co., Rockfield, IL) of Smith etal. (’85) using BSA as a standard. All measure-ments were made in triplicate and averaged.

RESULTSOptimal citrulline assay conditions

Mitochondrial citrulline synthesis was inverselyrelated to the concentration of succinate used inthe reaction medium between values of 0.1 and10 mM (Fig. 1). The peak rate of synthesis oc-curred at 0.1 mM, but the rates for 5 and 10 mMwere only slightly above those measured in thecomplete absence of succinate.

With 0.1 mM succinate, citrulline synthesisreached a maximum rate using 5 mM GLN(Fig. 2.).

Under these assay conditions, mitochondriafrom uncrowded toadfish produced citrulline in alinear fashion but very slowly and only to a slightdegree (Fig. 3A). There was no apparent differ-ence in either the rate of accumulation or the fi-nal amount of citrulline regardless of the amountof succinate used (0, 0.1, or 10 mM). The actualrate of synthesis declined over time (Fig. 3B). Forcrowded toadfish, mitochondrial citrulline synthe-sis was virtually undetectable when either 0 or10 mM succinate was used (Fig. 3C,D). For 0.1mM succinate, citrulline production was linearover 25 min with a disproportionate increase oc-curring over the last 5 min of the assay (Fig. 3C).The linear portion of citrulline production occurred


at a constant rate of synthesis of about 0.08 µmolgm–1 min–1 between 5 and 25 min (Fig. 3D); therate actually increased by nearly 2 fold over thefinal 5 min of the assay.

Mitochondrial CA activityMitochondria from all preparations contained

significant CA activity. Activity was not differentin mitochondria from uncrowded (31.8 ± 7.8 µmol

g–1 min–1, n = 5) vs. crowded (29.4 ± 6.7 µmol g–1

min–1, n = 8) fish. These values were slightly lowerbut in the general range of values previously re-ported for toadfish liver mitochondria CA activityusing very similar isolation and assay procedures(Walsh et al., ’89).

Mitrochondrial citrulline synthesisMitochondria from crowded fish synthesized cit-

rulline at a rate of about 0.12 µmol g–1 min–1; thiswas nearly 10-fold greater than the rate for mito-chondria from uncrowded fish (Fig. 4). Both CA in-hibitors, Az and Mz, reduced citrulline synthesis byapproximately 75%; treatment with QAS resultedin a slight but statistically insignificant reductionin citrulline production (P > 0.05, student’s t-test;Fig. 4). The addition of BCA to the reaction me-dium had no effect on citrulline synthesis (Fig. 4).

The use of 5 mM NaHCO3 appeared to producemaximal citrulline synthesis regardless of thepresence or absence of exogenous CA. For mito-chondria from crowded toadfish, citrulline synthe-sis was 0.04, 0.12, and 0.11 µmol g–1 min–1 for 0,5, and 25 mM HCO3

–, respectively, using 0.1 mMsuccinate and with no CA added to the reactionmedium (n = 2–3). There was no apparent differ-ence in citrulline synthesis for the same threeHCO3

– concentrations in the presence of exogenousCA (0.04, 0.13, and 0.08 µmol g–1 min–1, respec-tively, n = 2–3). Preliminary data indicate that,in the absence of succinate, HCO3

– concentrationcan have an effect on citrulline synthesis: the rateswere appoximately doubled at 5 mM NaHCO3 inthe presence or absence of exogenous CA (0.03,0.05, and 0.03 µmol g–1 min–1 for 0, 5, and 25 mMNaHCO3, respectively, n = 2).

Interestingly, mitochondria from crowded fish ex-posed to MS 222 had significantly depressed ratesof citrulline synthesis. MS 222 treatment resultedin an 80% inhibition of citrulline production (Fig. 4).

Mitochondrial glutamine uptakeMitochondrial GLN-derived radioactivity in-

creased in a near linear fashion over time from2.5 to 10 sec and then levelled off through 20 sec(Fig. 5), confirming 4 sec as a reproducible andshort enough time to be on the most linear por-tion of the curve for the subsequent experiments.However, given the slight displacement of the 2.5to 10 sec values from a direct line from the ori-gin, there is possibly an even faster uptake com-ponent which was not detected by the currentmethodology. Uptake rates as a function of GLNconcentration followed Michaelis-Menton kinetics,

Fig. 1. Citrulline synthesis from mitochondria isolatedfrom crowded toadfish vs succinate concentration in the re-action medium. Points are the average of triplicate measure-ments.

Fig. 2. Citrulline synthesis from mitochondria isolatedfrom crowded toadfish vs glutamine concentration in the re-action medium. All measurements made in the presence of0.1 mM succinate. Points are the average of triplicate mea-surements.


so Lineweaver-Burke transformations were usedto estimate Km and Vmax. Toadfish liver mito-chondria Vmax values (Table 1) were at the lowend of the range of those observed for rat kidneymitochondria (Goldstein and Boylan, ’78), and Kmvalues were in the range reported for toadfish liverGLN content in vivo (Walsh and Milligan, ’95).Km increased three-fold and Vmax increased four-fold in crowded toadfish, indicating that circum-stances that activate ureogenesis and ureotely alsoenhance mitochondrial transport capacity for thesubstrate glutamine.

DISCUSSIONA 10-fold increase in citrulline synthesis was

observed in mitochondria from crowded vs un-crowded toadfish, but this difference was not ap-

Fig. 3. Citrulline production over time in mitochondriaisolated from uncrowded (A) vs. crowded (C) toadfish, andthe rate of citrulline synthesis in mitochondria from un-crowded (B) vs. crowded (D) toadfish. Open circles = 0 mM

succinate; triangles = 0.1 mM succinate; squares = 10 mMsuccinate. All points are the average of triplicate measure-ments.

parent when the colorimetric assay was rununder conditions (notably high succinate concen-trations) used previously for a radioisotopic cit-rulline assay. Anderson and Walsh (’95), using theradioisotopic assay which measures incorporationof 14C-HCO3

– into an acid-stable fraction, reportedproduction of 0.35 µmol of citrulline over a 25 minincubation period for 0.75 mg mitochondrial pro-tein, using 10 mM succinate. That amount wassimilar to values reported here using 10 mM suc-cinate for both uncrowded and crowded toadfish(Fig. 3A,C). The use of 0.1 mM succinate (vs. 0 or10 mM) did not alter either the accumulation ofcitrulline in uncrowded toadfish or its rate of syn-thesis. Also, it was not possible to discern a dif-ference in citrulline synthesis between conditionsin which succinate was present or absent from


the assay, indicating that mitochondrial citrullineproduction in uncrowded toadfish is minimal, evenin the presence of high levels of GLN.

Toadfish hepatocyte urea synthesis has beenlinked to an induction of cytoplasmic glutaminesynthase (GSase; Walsh and Milligan, ’95; Hop-kins et al., ’95); however, it appears that there isalso another component of the ornithine-urea cyclethat is induced during the transition to urea-genesis. This is indicated by the results with cit-rulline production in mitochondria from crowdedtoadfish. In the presence of 0.1 mM succinate, cit-rulline production was about 10-fold higher than

in uncrowded fish (Fig. 4). However, citrulline syn-thesis appeared to be inhibited by higher succi-nate concentrations, the rate for 10 mM succinatebeing no different from that in the complete ab-sence of succinate (Fig. 3D). The inhibitory effectof succinate could be occurring either by a com-petitive inhibition of GLN transport into the mi-tochondria or by inhibiting one of the reactions inthe citrulline pathway. Although the mechanismwas not tested directly, the delay in the inhibi-tory effect (maximal inhibition takes about 15–20min to occur, Fig. 3B) suggests that succinate isworking on the reaction pathway via a slow accu-mulation within the intramitochondrial space.

Furthermore, even though high rates of citrul-line synthesis were observed using 0.1 mM succi-nate, the true optimal concentration for maximalcitrulline synthesis in vivo may be even lower.Both the accumulation of citrulline and its rateof synthesis doubled over the last 5 min of themeasurement (between 25 and 30 min; Fig. 3C,D),suggesting that citrulline synthesis increased oncea certain amount of the available succinate wasconsumed and its in vivo concentration decreasedbelow a critical point. This possibility deservesmore systematic examination.

This information suggests that the rate of cit-rulline production reported by Anderson andWalsh (’95) is very conservative. As such, actualin vivo rates of citrulline synthesis are probablywell above those necessary to support both ureaproduction in isolated hepatocytes and whole or-ganism rates of urea excretion (Barber and Walsh,’93). The radioisotopic assay may be sensitiveenough to detect small differences in citrullinesynthesis in the presence of 10 mM succinate, butthe colorimetric assay is not.

As proposed by Walsh (’97b), the stress-activa-tion of urea production in toadfish liver appearsto not only involve an increase in cytoplasmicglutamine production by GSase, but also a coor-dinated increase in the ability of the mitochon-dria to take up GLN so produced (Table 1). Atransient increase in plasma levels of cortisol, dur-

Fig. 4. Citrulline synthesis in isolated toadfish mitochon-dria. Bars represent the Mean ± SEM for the following con-ditions: UC = uncrowded control (n = 4); CC = crowded control(n = 10); CAz = crowded plus 1 mM acetazolamide (n = 5);CMz = crowded plus 1 mM methazolamide (n = 5); CCA =crowded plus 0.1 mg ml–1 bovine CA (n = 7); CQAS = crowdedplus 10 mM QAS (n = 4); CMS222 = crowded fish treatedwith MS 222 before mitochondrial isolation (n = 2).

Fig. 5. Plot of glutamine accumulation vs time in mito-chondria isolated from liver of crowded toadfish. Values aremean ± SEM (n = 3).

TABLE 1. Kinetic constants for glutamine uptake bymitochondria isolated from livers of uncrowded

or crowded toadfish1

VmaxCondition Km (mM) (nmoles mg protein–1 sec–1)

Uncrowded 7.21 ± 1.73 0.200 ± 0.023Crowded 22.24 ± 5.84* 0.887 ± 0.271*1Mean ± SEM, n = 5, where each pooled preparation is one sample.*Significantly different from uncrowded (P < 0.05, student’s t-test).


ing the initial 24 h of crowding, has been shownto be necessary for the increase in GSase (Hopkinset al., ’95). Whether the induction of GLN trans-port is also under the influence of cortisol, andwhether the induction of the enzyme and thetransport protein are coordinated or independentevents is not known at this time.

It is informative to attempt to place the kineticparameters for mitochondrial GLN uptake and cit-rulline synthesis in an in vivo context. First, thehepatic GLN content in toadfish rises from about2.7 to about 5 mmol Kg–1 upon confinement (Walshand Milligan, ’95). Interestingly, although the Kmfor mitochondrial uptake of GLN in uncrowdedtoadfish is above these in vivo concentrations,there is still an adaptive increase in both Km andVmax of GLN transport, such that the transportsystem continues to have a great deal of reservecapacity for GLN, and would likely not be satu-rated in vivo. Indeed, the increase in transportcapacity reported here is in close agreement withthe three-to-four fold increase in cytoplasmicGSase (Walsh and Milligan, ’95). Notably whentransport rates for GLN are converted to theunits of µmol g–1 min–1 used for citrulline pro-duction (by multiplication of the values in Table1 by a factor of 720), it is clear that transportcapacity exceeds the rate of mitochondrial cit-rulline production by approximately five-fold.Furthermore, since the rate of mitochondrialcitrulline production saturates at 5 mM GLN,the maximum concentration observed in vivo,these results would appear to rule out GLNtransport as being rate-limiting to urea produc-tion. Anderson and Walsh (’95) concluded thaturea production was limited by nitrogen avail-ability, and the results presented here supporttheir original proposal that the rates of ureasynthesis are regulated by a balance betweenGLN production by cytoplasmic GSase and con-sumption by mitochondrial CPSase III.

Our results also indicate that CPSase III is de-pendent on a second enzyme for substrate supply,namely intramitochondrial CA which suppliesHCO3

–. As was reported for urea synthesis in in-tact hepatocytes (Walsh et al., ’89), carbonic an-hydrase (CA) is essential for normal rates ofcitrulline synthesis in isolated mitochondria. Bothacetazolamide (Az) and methazolamide (Mz), CAinhibitors which permeate into the mitochondrialmatrix, inhibit citrulline production by about 70–80%. This is very similar to the degree of inhibi-tion for citrulline synthesis in isolated guinea pigliver mitochondria (Dodgson et al., ’83), and for

urea synthesis in isolated perfused rat and hu-man liver (Haussinger and Gerok, ’85; Haussingeret al., ’86), and in isolated hepatocytes from theliver of guinea pigs (Dodgson and Forster, ’86) andtoadfish (Walsh et al., ’89). QAS, a CA inhibitorthat is excluded from the mitochondrial matrix,had no effect on citrulline synthesis. Furthermore,the addition of bovine CA to the reaction mediumdid not increase citrulline production. These re-sults support the role of mitochondrial matrix CAin citrulline synthesis exclusive of any support-ing function of cytoplasmic CA activity. It does notappear that mitochondrial synthesis is HCO3

– lim-ited even under conditions of peak urea produc-tion, and there is no evidence indicating that CO2transport from the cytoplasm into the mitochon-dria is necessary to maintain peak citrullinesynthesis. Citrulline synthesis appears to be de-pendent on intramitochondrial CO2 productionand on the activity of matrix CA to maintainchemical equilibrium between CO2 and HCO3

– inorder to keep CPSase III supplied with substrate.Mitochondrial CA activity is not induced incrowded toadfish, indicating that the enzyme isthere in excess, a condition that is considered al-most universal for CA activity, regardless of itslocalization or function.

Treatment of crowded toadfish with MS 222prior to use in an experiment resulted in severelydepressed mitochondrial citrulline synthesis. Thiscould be a result of the anesthetic working at twopossible levels of inhibition. First, MS 222 isknown to directly inhibit CA activity (Christensenand Tucker, ’76); the compound has been shownto cause an approximate 20% inhibition of red cellCA activity in catfish. Given the very high totalconcentration of CA in catfish blood (Henry et al.,’88), 20% is a significant amount of inhibition.Since mitochondrial CA activity is much lowerthan that in red cells, it is quite possible thatenough MS 222 could have permeated into themitochondria to cause a great enough degree ofenzyme inhibition to result in depressed citrul-line synthesis. A second possibility could be thatMS 222 could be interfering with mitrochondrialGLN uptake. MS 222, like other anesthetics, prob-ably acts as a general membrane stabilizing agent,and this could have impacted on a number oftransport events, including the uptake of GLN.Regardless of its mechanism of action, however,the effects are clear, and it becomes important toavoid the use of anesthetics such as MS 222 instudies involving the measurement of metabolicprocesses.


ACKNOWLEDGMENTSThis work was supported by NSF IBN 93-04844

to R.P.H. and to NSF IBN 95-07239 to P.J.W. Wethank Jimbo Luznar for collecting the toadfish andJohn Paupe for technical assistance above and be-yond the call of duty.

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mitochondrial citrulline synthesis in the ureagenic toadfish,opsanus beta, is dependent on carbonic...

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