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JEZ 867
Mitochondrial Citrulline Synthesis in the Ureagenic
Toadfish, Opsanus beta, Is Dependent on Carbonic
Anhydrase Activity and Glutamine Transport
Department of Zoology and Wildlife Science, Auburn University, Auburn,
Alabama 36849-5414
Division of Marine Biology and Fisheries, Rosenstiel School of Marine and
Atmospheric Science, University of Miami, Miami, Florida 33149-1098
Mitochondria isolated from the liver of the Gulf toadfish Opsanus beta produce
scitrulline in the presence of 5 mM glutamine. Citrulline production was inversely related to
succinate 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, mitochondrial
citrulline production increases by approximately 10-fold. Citrulline synthesis is dependent on
intramitochondrial carbonic anhydrase (CA) activity, being inhibited by about 75% by both acetazolamide and methazolamide. The addition of exogenous CA did not increase mitochondrial citrulline production. Anesthetizing the fish with MS 222 prior to mitochondrial isolation resulted
in the near elimination of the capacity for citrulline synthesis. Mitochondria were also shown to
possess an inducible glutamine transport system. The Vmax for glutamine uptake increased threefold and the Km increased four-fold in ureagenic vs. ammoniogenic toadfish. The transport system is the second labile component of the overall ornithine-urea cycle to be identified, and it
provides a link between the production of glutamine via cytoplasmic glutamine synthetase and
its 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 the
few teleost fish that has the capacity to become
ureotelic on a facultative basis. Under conditions
of minimal stress (i.e., individual fish isolated in
large holding tanks) toadfish are primarily ammonotelic, excreting 61–80% of their total nitrogen as ammonia, depending on their nutritional
status (starved vs. fed, Walsh and Milligan, ’95).
However, when animals are either crowded together or confined individually in small, restricted
chambers, their pattern of nitrogen excretion is
altered such that urea becomes the predominant
excretory product, accounting for 90% of the total
nitrogen excreted (Walsh et al., ’94; Walsh and
Milligan, ’95).
Unlike ammonia, which is excreted in a continuous, uniform fashion, urea excretion in toadfish occurs in a periodic manner; most of the
excreted urea is released in a single pulse of less
than 3 h duration aproximately once every 24 h
(Walsh et al., ’90; Wood et al., ’95). The pulse is
released from the head region and is believed to
originate from the gills (Wood et al., ’95).
While the properties of urea excretion have been
extensively characterized in this species, much
less is known about the mechanism, control, and
potential lability of urea synthesis. Toadfish are
known to possess a functional and complete hepatic ornithine-urea cycle with a glutamine-dependent carbamoyl phosphate synthesis (CPSase III;
Mommsen and Walsh, ’89; Anderson and Walsh,
’95). The transition from ammonotelism to ureotelism involves the induction of the enzyme
glutamine synthetase (GSase) in the liver which
is 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 ammonia-nitrogen is channeled into the ornithine-urea
cycle as glutamine (Walsh et al., ’90; Hopkins et
al., ’95; Walsh and Milligan, ’95; Anderson and
Walsh, ’95). Once the transition to ureotelism is
complete, urea synthesis appears to occur at a constant rate with only urea excretion being pulsa-
*Correspondence to: Raymond P. Henry, Dept. of Zoology and Wildlife Science, 101 Cary Hall, Auburn University, Auburn, AL 368495414. E-mail:
tile (Wood et al., ’97). However, it is not known if
other components of the urea pathway are altered
as well during that transition in order to achieve
higher rates of synthesis. One potential point of
control could occur between GSase and CPSase
III. There are both cytoplasmic and mitochondrial
isozymes of GSase (Walsh, ’97a), and it is the
cytoplasmic isozyme which is induced (Milligan
and Walsh, ’95). Because CPSase III is mitochondrial, it has been speculated that one point
of control could occur via the regulation of
glutamine transport across the mitochondrial
membrane (Walsh, ’97b).
Very little is known about the intramitochondrial
aspects of urea synthesis in toadfish as well, although it is clear that they are capable of citrulline
synthesis under some circumstances (Anderson and
Walsh, ’95). Studies on isolated hepatocytes from
toadfish have documented significant levels of urea
synthesis, showing that CPSase III is dependent
on GSase for nitrogen and on carbonic anhydrase
(CA) for HCO3– (Walsh et al., ’89). Inhibition of total hepatocyte CA activity by acetazolamide reduced
urea synthesis by a maximum of about 70%, but
since both the cytoplasm and mitochondria contain
CA activity, it could not be determined if CA in one
or both subcellular compartments was necessary for
HCO3– supply to CPSase III. Some evidence from
mammalian systems exists suggesting that CO2 produced endogenously within the mitochondria may
not be sufficient to meet the metabolic carbon demand for urea synthesis. The rate of urea synthesis was reduced by about 60% in isolated guinea
pig hepatocytes when external CO2 was reduced
from 25 mM to zero (Dodgson and Forster, ’86). Furthermore, Tomera et al. (’82) reported that the rate
of 14C fixation from NaH14CO3 into urea in perfused
rat liver was much higher than the net rate of CO2
production. This suggests that cytoplasmic CO2 may
be diffusing into the mitochondrial matrix to support urea synthesis. If so, cytoplasmic CA may be
important in maintaining the transport of CO2
across the mitochondrial membrane (reviewed by
Gros and Dodgson, ’88; Henry, ’96).
This investigation reports on the mitochondrial
component of urea synthesis in the toadfish liver.
Citrulline synthesis was used as an indicator of
mitochondrial nitrogen metabolism in order to elucidate the factors influencing the induction and
regulation of urea synthesis.
Collection and maintenance of animals
Adult Gulf toadfish Opsanus beta (Goode and
Bean) were collected with a roller trawl in Bis-
cayne Bay, Florida, by commercial shrimpers
during April and May, 1996, for the metabolic experiments, and between July and October, 1996,
for the transport experiments. Fish were housed
in 80 L aquaria with flowing aerated seawater
(detailed procedures given by Walsh and Milligan,
’95). Individuals were fed shrimp but were starved
for a period of 72 h before being used in an experiment.
Fish were initially held under conditions of low
population density (uncrowded). High population
density (crowding) was used to initiate ureogenesis: fish were transferred to 6 L plastic tubs
(30 cm long x 25 cm wide × 10 cm high) with flowing seawater such that there were four to six individuals per tub (Walsh et al., ’94). Fish were
kept under these conditions for a minimum of 4
days before being used.
Mitochondria preparation
Fish were killed by a blow to the head either
with or without having first been lightly anesthetized in MS 222 (tricaine methanesulfonate
buffered with NaHCO3). The liver was dissected
out, and blood vessels and connective tissue were
cleaned away. Fish were used individually when
large enough to yield 1.5 gm or more of liver; otherwise, livers for two to three fish were pooled.
Mitochondria were prepared according to the
method outlined by Anderson and Walsh (’95).
Briefly, the liver was suspended in 5.5 volumes of
homogenization buffer (250 mM sucrose, 0.5 mM
EGTA, 5 mM K2HPO4, 30 mM HEPES, pH = 7.4
at 22°C), minced with scissors, and homogenized
using a motor-driven teflon-glass homogenizer
(four to six passes). The homogenate was centrifuged for 10 min at 750 × g (SS-34 rotor, Sorvall
RC5-B superspeed) at 4°C. The supernatant was
saved and centrifuged at 14,500 g for 10 min to
produce the initial mitochondrial pellet. This was
washed once in nine volumes of homogenization
buffer and recentrifuged as above. The final pellet was resuspended in a volume of homogenization buffer plus bovine serum albumin (BSA, 5
mg ml–1), made fresh daily, equivalent to 0.3 ml
g–1 of starting weight of liver.
Carbonic anhydrase activity
A 50 µl sample from each preparation was
saved, frozen at –80°C, and shipped to Auburn
on dry ice for analysis of CA activity. Samples
were thawed on ice, sonicated, and measured
using the electrometric delta pH assay (Henry,
’91). CA activity was reported on the basis of
fresh weight of tissue.
Citrulline assay
The reaction medium for the analysis of mitochondrial citrulline synthesis was slightly
modified from that used by Anderson and Walsh
(’95; see below). This was made fresh daily and
consisted of the following: 88 mM sucrose, 0.175
mM EGTA, 6 mM K2HPO4, 90 mM KCl, 5 mM
NaHCO3, 2 mM MgCl2, 0.15 mM ATP, 10 mM ornithine, 0.1 mM succinate, and 36 mM HEPES,
pH = 7.40 at 22°C. Seventy five microliters of the
mitochondrial suspension were added to 500 µl of
the reaction medium in a 1.5 ml microcentrifuge
tube; this was allowed to equilibrate to room temperature (22°C) for 20 min. The reaction was
started by adding 50 µl of a 50 mM glutamine
(GLN) stock solution and gently mixing. After 30
min the reaction was stopped by the addition of
10 µl of 70% perchloric acid (PCA). The tubes were
centrifuged for 30 sec at 10,000 g, and the supernatant was assayed for citrulline concentration.
The citrulline assay reagent consisted of a 1:1
(v:v) mixture of antipyrene (2,3-dimethyl-1-phenyl-3pyrazolin-5-One, 200 mg in 94 ml H2O, 6 ml
concentrated H2SO4, stored in a dark bottle at
room temperature) and diacetyl monoxime (900
mg 2,3 butanedione monoxime in 95 ml H2O, 5
ml glacial acetic acid, stored refrigerated in a dark
bottle) made fresh before use. One ml of this reagent was added to 50 µl of sample in a glass
tube, mixed, loosely capped with a marble, and
boiled in the dark for 30 min. The tubes were then
cooled in an ice bath in the dark. Samples were
transferred to semi-micro cuvettes, and absorbance was read at 464 nm (Perkin-Elmer Lambda
2). The increase in citrulline concentration was
determined by comparison against a standard
curve of 0 to 50 µM citrulline. All chemicals were
purchased from Sigma (St. Louis, MO) and were
reagent grade.
Experimental protocol
Citrulline synthesis was measured for mitochondria from toadfish that had been held either under uncrowded conditions for at least 7 days or
crowded conditions for a minimum of 4 days. Because the initial measurements of the rates of citrulline synthesis were very low in both groups,
we reinvestigated the assay conditions used previously (Anderson and Walsh, ’95). Both the linearity of citrulline accumulation over time and the
rate of citrulline synthesis were tested against
varying concentrations of GLN and succinate. Furthermore, even after the optimal assay conditions
were established, citrulline synthesis within a
treatment group of fish was at times highly variable, with some individual preparations showing
no apparent citrulline synthesis. In order to determine if this was a result of the anesthetic (MS
222) depressing one or more components of the
citrulline pathway, synthesis was measured on
mitochondria prepared from fish that were either
killed after being anesthetized using MS 222 or
killed immediately without anesthesia.
For the remainder of the experiments, toadfish
were killed without prior exposure to MS 222. Mitochondria from these preparations were used to
determine if citrulline synthesis was dependent
on mitochondrial and/or cytoplasmic CA activity.
The CA inhibitors acetazolamide (Az) and methazolamide (Mz) were used to inhibit total CA activity (within the mitochondrial matrix and any
residual CA activity in the general reaction medium). Stock solutions (10 mM) were made up in
reaction medium; these were added to the final
mitochondrial suspensions (also in reaction medium) in a 1:10 dilution to give a working concentration of 1 mM. The mitochondria were allowed
to incubate at room temperature for 30 min, and
the reaction was started by the addition of GLN.
Another CA inhibitor, quaternary ammonium sulfanilamide (QAS), was used to selectively inhibit
CA activity in the reaction medium, leaving CA
within the matrix functional (Henry, ’87). A stock
solutin of QAS in reaction medium (100 mM) was
diluted 1:10 for a final working concentration of
10 mM; mitochondria were incubated for 30 min
before GLN addition.
In order to determine the potential importance
of extra-mitochondrial (i.e., cytoplasmic) CO2 in
citrulline synthesis, exogenous CA activity was
added to the reaction medium. Purified bovine red
cell CA (BCA) was added to the reaction medium
to a final concentration of 0.1 mg ml–1. In a separate set of experiments, a series of HCO3– concentrations in the reaction medium (0, 5, and 25 mM)
was used in order to determine the sensitivity of
citrulline production to external CO2 concentrations. These measurements were carried out in
the presence of 0.1 mM succinate with and without BCA, and in the absence of succinate with and
without BCA.
For each experiment, a blank consisting of the
final mitochondrial suspension without the addition of GLN was carried out to control for preexisting levels of citrulline.
Mitochondrial glutamine transport
Mitochondria were isolated as above with slight
modification. Livers from four to five fish were
pooled to give 3.5 to 5 g starting weight. The homogenization medium consisted of 250 mM sucrose, 0.5 mM EGTA and 30 mM HEPES, pH =
7.40 at 22°C. The washed mitochondrial pellet was
suspended to 0.15 ml g–1 original starting weight
using homogenization buffer plus 5 mg ml–1 BSA
and kept on ice until use.
The rapid mixing/rapid filtration method of
Goldstein and Boylan (’78) was used to measure
the rate of GLN uptake. In general, 50 µl of the
mitochondrial suspension was drawn into a 100
µl Hamilton syringe followed by 50 µl of mitochondrial transport medium (99 mM sucrose, 0.175
mM EGTA, 90 mM KCl, 2 mM MgCl2, 36 mM
HEPES, pH = 7.40 at 22°C plus the metabolic inhibitors 2 µg ml–1 rotenone and 1.5 µg ml–1 antimycin A). The contents were ejected into a 1.5 ml
microcentrifuge tube and quickly withdrawn again
to facilitate mixing of the mitochondria with the
transport medium. The mixture was allowed to
stand for 1 min prior to injection into the uptake
vessel in order to facilitate the action of the metabolic inhibitors. A second syringe contained 100
µl of the mitochondrial transport medium plus 0.2
µCi of L-[14C(U)]-GLN (200 mCi mmol–1, NEC-451,
DuPont NEN) and twice the appropriate final concentration of unlabeled GLN. The first experimental series involved a fixed concentration of 0.2 mM
GLN and variable uptake times between 2.5 and
20 sec to determine the appropriate time to use
for kinetic measurements. The second experimental series used variable final GLN concentrations
between 0.2 and 5 mM and a fixed uptake time of
4 sec. For sucrose space determinations, 0.2 µCi
of [14C(U)]-sucrose (4.5 mCi mmol–1, NEC-100, DuPont NEN) was substituted for GLN in the transport medium. Briefly, the procedure involved the
very rapid and simultaneous injection of both syringes into a small mixing chamber, the bottom
of which is fitted with a Millipore filter supported
on a wire mesh (see Goldstein and Boylan, ’78 for
details of construction of the apparatus). Immediately after mixing, vacuum was applied to the
bottom of the chamber, trapping the mitochondria
on the filter; the filter was then washed with 10
ml of isolation medium containing no substrate.
The entire procedure was completed within 2.5
sec in our hands. The filter was then rapidly removed and placed in 10 ml Ecolume (ICN) and
counted in a Tracor Analytic LSC. Uptake rates
of GLN were calculated according to the equations
of Goldstein and Boylan (’78) as follows:
matrix GLN dpms = [GLNf] –
where GLNf = the total GLN dpms of the filter,
SUCROSEf = the dpms on the filter in the sucrose incubations, SUCROSEm = the total sucrose
dpms in the medium, and GLNm = the GLN dpms
in the medium. And:
nmol GLN matrix = matrix GLN
dpms/GLN specific activity
where specific activity is expressed as dpms
nmol–1. Uptakes were then divided by 4 sec and normalized on a per mg mitochondrial protein basis.
Protein was determined on an aliquot of mitochondrial suspension by sonicating 1:1 in 50 mM
HEPES with 0.1% Triton X-100 followed by assay according to the bicinchoninic acid method
(Pierce Chemical Co., Rockfield, IL) of Smith et
al. (’85) using BSA as a standard. All measurements were made in triplicate and averaged.
Optimal citrulline assay conditions
Mitochondrial citrulline synthesis was inversely
related to the concentration of succinate used in
the reaction medium between values of 0.1 and
10 mM (Fig. 1). The peak rate of synthesis occurred at 0.1 mM, but the rates for 5 and 10 mM
were only slightly above those measured in the
complete absence of succinate.
With 0.1 mM succinate, citrulline synthesis
reached a maximum rate using 5 mM GLN
(Fig. 2.).
Under these assay conditions, mitochondria
from uncrowded toadfish produced citrulline in a
linear fashion but very slowly and only to a slight
degree (Fig. 3A). There was no apparent difference in either the rate of accumulation or the final amount of citrulline regardless of the amount
of succinate used (0, 0.1, or 10 mM). The actual
rate of synthesis declined over time (Fig. 3B). For
crowded toadfish, mitochondrial citrulline synthesis was virtually undetectable when either 0 or
10 mM succinate was used (Fig. 3C,D). For 0.1
mM succinate, citrulline production was linear
over 25 min with a disproportionate increase occurring over the last 5 min of the assay (Fig. 3C).
The linear portion of citrulline production occurred
g–1 min–1, n = 5) vs. crowded (29.4 ± 6.7 µmol g–1
min–1, n = 8) fish. These values were slightly lower
but in the general range of values previously reported for toadfish liver mitochondria CA activity
using very similar isolation and assay procedures
(Walsh et al., ’89).
Mitrochondrial citrulline synthesis
Fig. 1. Citrulline synthesis from mitochondria isolated
from crowded toadfish vs succinate concentration in the reaction medium. Points are the average of triplicate measurements.
at a constant rate of synthesis of about 0.08 µmol
gm–1 min–1 between 5 and 25 min (Fig. 3D); the
rate actually increased by nearly 2 fold over the
final 5 min of the assay.
Mitochondrial CA activity
Mitochondria from all preparations contained
significant CA activity. Activity was not different
in mitochondria from uncrowded (31.8 ± 7.8 µmol
Mitochondria from crowded fish synthesized citrulline at a rate of about 0.12 µmol g–1 min–1; this
was nearly 10-fold greater than the rate for mitochondria from uncrowded fish (Fig. 4). Both CA inhibitors, Az and Mz, reduced citrulline synthesis by
approximately 75%; treatment with QAS resulted
in a slight but statistically insignificant reduction
in citrulline production (P > 0.05, student’s t-test;
Fig. 4). The addition of BCA to the reaction medium had no effect on citrulline synthesis (Fig. 4).
The use of 5 mM NaHCO3 appeared to produce
maximal citrulline synthesis regardless of the
presence or absence of exogenous CA. For mitochondria from crowded toadfish, citrulline synthesis was 0.04, 0.12, and 0.11 µmol g–1 min–1 for 0,
5, and 25 mM HCO3–, respectively, using 0.1 mM
succinate and with no CA added to the reaction
medium (n = 2–3). There was no apparent difference in citrulline synthesis for the same three
HCO3– concentrations in the presence of exogenous
CA (0.04, 0.13, and 0.08 µmol g–1 min–1, respectively, n = 2–3). Preliminary data indicate that,
in the absence of succinate, HCO3– concentration
can have an effect on citrulline synthesis: the rates
were appoximately doubled at 5 mM NaHCO3 in
the presence or absence of exogenous CA (0.03,
0.05, and 0.03 µmol g–1 min–1 for 0, 5, and 25 mM
NaHCO3, respectively, n = 2).
Interestingly, mitochondria from crowded fish exposed to MS 222 had significantly depressed rates
of citrulline synthesis. MS 222 treatment resulted
in an 80% inhibition of citrulline production (Fig. 4).
Mitochondrial glutamine uptake
Fig. 2. Citrulline synthesis from mitochondria isolated
from crowded toadfish vs glutamine concentration in the reaction medium. All measurements made in the presence of
0.1 mM succinate. Points are the average of triplicate measurements.
Mitochondrial GLN-derived radioactivity increased in a near linear fashion over time from
2.5 to 10 sec and then levelled off through 20 sec
(Fig. 5), confirming 4 sec as a reproducible and
short enough time to be on the most linear portion of the curve for the subsequent experiments.
However, given the slight displacement of the 2.5
to 10 sec values from a direct line from the origin, there is possibly an even faster uptake component which was not detected by the current
methodology. Uptake rates as a function of GLN
concentration followed Michaelis-Menton kinetics,
Fig. 3. Citrulline production over time in mitochondria
isolated from uncrowded (A) vs. crowded (C) toadfish, and
the rate of citrulline synthesis in mitochondria from uncrowded (B) vs. crowded (D) toadfish. Open circles = 0 mM
succinate; triangles = 0.1 mM succinate; squares = 10 mM
succinate. All points are the average of triplicate measurements.
so Lineweaver-Burke transformations were used
to estimate Km and Vmax. Toadfish liver mitochondria Vmax values (Table 1) were at the low
end of the range of those observed for rat kidney
mitochondria (Goldstein and Boylan, ’78), and Km
values were in the range reported for toadfish liver
GLN content in vivo (Walsh and Milligan, ’95).
Km increased three-fold and Vmax increased fourfold in crowded toadfish, indicating that circumstances that activate ureogenesis and ureotely also
enhance mitochondrial transport capacity for the
substrate glutamine.
parent when the colorimetric assay was run
under conditions (notably high succinate concentrations) used previously for a radioisotopic citrulline assay. Anderson and Walsh (’95), using the
radioisotopic assay which measures incorporation
of 14C-HCO3– into an acid-stable fraction, reported
production of 0.35 µmol of citrulline over a 25 min
incubation period for 0.75 mg mitochondrial protein, using 10 mM succinate. That amount was
similar to values reported here using 10 mM succinate for both uncrowded and crowded toadfish
(Fig. 3A,C). The use of 0.1 mM succinate (vs. 0 or
10 mM) did not alter either the accumulation of
citrulline in uncrowded toadfish or its rate of synthesis. Also, it was not possible to discern a difference in citrulline synthesis between conditions
in which succinate was present or absent from
A 10-fold increase in citrulline synthesis was
observed in mitochondria from crowded vs uncrowded toadfish, but this difference was not ap-
TABLE 1. Kinetic constants for glutamine uptake by
mitochondria isolated from livers of uncrowded
or crowded toadfish1
Km (mM)
7.21 ± 1.73
22.24 ± 5.84*
(nmoles mg protein–1 sec–1)
0.200 ± 0.023
0.887 ± 0.271*
Mean ± SEM, n = 5, where each pooled preparation is one sample.
*Significantly different from uncrowded (P < 0.05, student’s t-test).
Fig. 4. Citrulline synthesis in isolated toadfish mitochondria. Bars represent the Mean ± SEM for the following conditions: 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 = crowded
plus 10 mM QAS (n = 4); CMS222 = crowded fish treated
with MS 222 before mitochondrial isolation (n = 2).
the assay, indicating that mitochondrial citrulline
production in uncrowded toadfish is minimal, even
in the presence of high levels of GLN.
Toadfish hepatocyte urea synthesis has been
linked to an induction of cytoplasmic glutamine
synthase (GSase; Walsh and Milligan, ’95; Hopkins et al., ’95); however, it appears that there is
also another component of the ornithine-urea cycle
that is induced during the transition to ureagenesis. This is indicated by the results with citrulline production in mitochondria from crowded
toadfish. In the presence of 0.1 mM succinate, citrulline production was about 10-fold higher than
Fig. 5. Plot of glutamine accumulation vs time in mitochondria isolated from liver of crowded toadfish. Values are
mean ± SEM (n = 3).
in uncrowded fish (Fig. 4). However, citrulline synthesis appeared to be inhibited by higher succinate concentrations, the rate for 10 mM succinate
being no different from that in the complete absence of succinate (Fig. 3D). The inhibitory effect
of succinate could be occurring either by a competitive inhibition of GLN transport into the mitochondria or by inhibiting one of the reactions in
the citrulline pathway. Although the mechanism
was not tested directly, the delay in the inhibitory effect (maximal inhibition takes about 15–20
min to occur, Fig. 3B) suggests that succinate is
working on the reaction pathway via a slow accumulation within the intramitochondrial space.
Furthermore, even though high rates of citrulline synthesis were observed using 0.1 mM succinate, the true optimal concentration for maximal
citrulline synthesis in vivo may be even lower.
Both the accumulation of citrulline and its rate
of synthesis doubled over the last 5 min of the
measurement (between 25 and 30 min; Fig. 3C,D),
suggesting that citrulline synthesis increased once
a certain amount of the available succinate was
consumed and its in vivo concentration decreased
below a critical point. This possibility deserves
more systematic examination.
This information suggests that the rate of citrulline production reported by Anderson and
Walsh (’95) is very conservative. As such, actual
in vivo rates of citrulline synthesis are probably
well above those necessary to support both urea
production in isolated hepatocytes and whole organism rates of urea excretion (Barber and Walsh,
’93). The radioisotopic assay may be sensitive
enough to detect small differences in citrulline
synthesis in the presence of 10 mM succinate, but
the colorimetric assay is not.
As proposed by Walsh (’97b), the stress-activation of urea production in toadfish liver appears
to not only involve an increase in cytoplasmic
glutamine production by GSase, but also a coordinated increase in the ability of the mitochondria to take up GLN so produced (Table 1). A
transient increase in plasma levels of cortisol, dur-
ing the initial 24 h of crowding, has been shown
to be necessary for the increase in GSase (Hopkins
et al., ’95). Whether the induction of GLN transport is also under the influence of cortisol, and
whether the induction of the enzyme and the
transport protein are coordinated or independent
events is not known at this time.
It is informative to attempt to place the kinetic
parameters for mitochondrial GLN uptake and citrulline synthesis in an in vivo context. First, the
hepatic GLN content in toadfish rises from about
2.7 to about 5 mmol Kg–1 upon confinement (Walsh
and Milligan, ’95). Interestingly, although the Km
for mitochondrial uptake of GLN in uncrowded
toadfish is above these in vivo concentrations,
there is still an adaptive increase in both Km and
Vmax of GLN transport, such that the transport
system continues to have a great deal of reserve
capacity for GLN, and would likely not be saturated in vivo. Indeed, the increase in transport
capacity reported here is in close agreement with
the three-to-four fold increase in cytoplasmic
GSase (Walsh and Milligan, ’95). Notably when
transport rates for GLN are converted to the
units of µmol g–1 min–1 used for citrulline production (by multiplication of the values in Table
1 by a factor of 720), it is clear that transport
capacity exceeds the rate of mitochondrial citrulline production by approximately five-fold.
Furthermore, since the rate of mitochondrial
citrulline production saturates at 5 mM GLN,
the maximum concentration observed in vivo,
these results would appear to rule out GLN
transport as being rate-limiting to urea production. Anderson and Walsh (’95) concluded that
urea production was limited by nitrogen availability, and the results presented here support
their original proposal that the rates of urea
synthesis are regulated by a balance between
GLN production by cytoplasmic GSase and consumption by mitochondrial CPSase III.
Our results also indicate that CPSase III is dependent on a second enzyme for substrate supply,
namely intramitochondrial CA which supplies
HCO3–. As was reported for urea synthesis in intact hepatocytes (Walsh et al., ’89), carbonic anhydrase (CA) is essential for normal rates of
citrulline synthesis in isolated mitochondria. Both
acetazolamide (Az) and methazolamide (Mz), CA
inhibitors which permeate into the mitochondrial
matrix, inhibit citrulline production by about 70–
80%. This is very similar to the degree of inhibition for citrulline synthesis in isolated guinea pig
liver mitochondria (Dodgson et al., ’83), and for
urea synthesis in isolated perfused rat and human liver (Haussinger and Gerok, ’85; Haussinger
et al., ’86), and in isolated hepatocytes from the
liver of guinea pigs (Dodgson and Forster, ’86) and
toadfish (Walsh et al., ’89). QAS, a CA inhibitor
that is excluded from the mitochondrial matrix,
had no effect on citrulline synthesis. Furthermore,
the addition of bovine CA to the reaction medium
did not increase citrulline production. These results support the role of mitochondrial matrix CA
in citrulline synthesis exclusive of any supporting function of cytoplasmic CA activity. It does not
appear that mitochondrial synthesis is HCO3– limited even under conditions of peak urea production, and there is no evidence indicating that CO2
transport from the cytoplasm into the mitochondria is necessary to maintain peak citrulline
synthesis. Citrulline synthesis appears to be dependent on intramitochondrial CO2 production
and on the activity of matrix CA to maintain
chemical equilibrium between CO2 and HCO3– in
order to keep CPSase III supplied with substrate.
Mitochondrial CA activity is not induced in
crowded toadfish, indicating that the enzyme is
there in excess, a condition that is considered almost universal for CA activity, regardless of its
localization or function.
Treatment of crowded toadfish with MS 222
prior to use in an experiment resulted in severely
depressed mitochondrial citrulline synthesis. This
could be a result of the anesthetic working at two
possible levels of inhibition. First, MS 222 is
known to directly inhibit CA activity (Christensen
and Tucker, ’76); the compound has been shown
to cause an approximate 20% inhibition of red cell
CA activity in catfish. Given the very high total
concentration of CA in catfish blood (Henry et al.,
’88), 20% is a significant amount of inhibition.
Since mitochondrial CA activity is much lower
than that in red cells, it is quite possible that
enough MS 222 could have permeated into the
mitochondria to cause a great enough degree of
enzyme inhibition to result in depressed citrulline synthesis. A second possibility could be that
MS 222 could be interfering with mitrochondrial
GLN uptake. MS 222, like other anesthetics, probably acts as a general membrane stabilizing agent,
and this could have impacted on a number of
transport events, including the uptake of GLN.
Regardless of its mechanism of action, however,
the effects are clear, and it becomes important to
avoid the use of anesthetics such as MS 222 in
studies involving the measurement of metabolic
This work was supported by NSF IBN 93-04844
to R.P.H. and to NSF IBN 95-07239 to P.J.W. We
thank Jimbo Luznar for collecting the toadfish and
John Paupe for technical assistance above and beyond the call of duty.
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