Vol. 283, Issue 3, R598-R603, September 2002
Comparison of the effects of ammonia on brain mitochondrial
function in rats and gulf toadfish
Clémence M.
Veauvy1,
Yuxiang
Wang1,2,
Patrick J.
Walsh1, and
Miguel A.
Pérez-Pinzón1,3
1 National Institute of Environmental Health
Sciences Marine and Freshwater Biomedical Science Center, Division
of Marine Biology and Fisheries, Rosenstiel School of Marine and
Atmospheric Science, University of Miami 33149;
3 Department of Neurology and Neuroscience, School
of Medicine, University of Miami, Miami, Florida 33131; and
2 Department of Biology, Queen's University, Kingston,
Ontario, K7L 3N6 Canada
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ABSTRACT |
We compared the effect of hyperammonemia
on NADH levels in brain slices and on the rate of oxygen consumption
from isolated nonsynaptic brain mitochondria in ammonia-sensitive
Wistar rats with that in ammonia-tolerant gulf toadfish (Opsanus
beta). The NADH content was significantly decreased (12% less
than control after 45 min with 1 mM NH4Cl) in rat brain
slices, but it was not affected in brain slices from toadfish (with
both 1 and 6 mM NH4Cl). The rates of oxygen consumption of
different sets of enzymes of the electron transport chain (ETC;
complexes I, II, III, and IV; II, III, and IV; and IV alone) were
unaltered by hyperammonemic conditions in isolated nonsynaptic
mitochondria from either rats or toadfish. These results lead us to
conclude that the differing effects of ammonia on NADH levels in rat
and toadfish brain slices must be due to aspects other than the direct effects of ammonia on enzymes of the ETC. Additionally, because these
effects were seen in vitro, our studies enabled us to rule out the
possibility that effects of ammonia on metabolism were via indirect
systemic effects. These results are discussed in the context of current
views on mechanisms of central nervous system damage in hyperammonemic states.
hepatic encephalopathy; hyperammonemia; NADH; mitochondria; Opsanus beta; glutamine metabolism
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INTRODUCTION |
MAMMALS IN GENERAL
are physiologically sensitive to increases in the concentration of
ammonia in the body. In particular, excess brain
ammonia1 (i.e., as low as 500 to 1,000 µmol/kg tissue wt) caused by liver failure (e.g., hepatic
encephalopathy) or inborn errors in urea metabolism or by injection of
ammonia in experimental animals leads to brain neuropathies. A general
sequence of symptoms progresses from altered sleep patterns to muscular
incoordination, stupor, coma, and death, at a rate that is dependent on
the extent and rate of ammonia intoxication; a lethal dose of ammonia
can cause symptoms within minutes [reviewed by Cooper and Plum
(6) and more recently by Hazell and Butterworth
(10)]. Much earlier literature focuses on the causes of
these neurological symptoms being related to "cerebral energy
failure." Indeed, in the early stages of hyperammonemia, brain
metabolic rate decreases, with precipitous drops in whole brain
creatine phosphate levels, whereas whole brain ATP content falls later
in the onset of disease. Because the decline in whole brain ATP follows
severe neurological impairment, many have argued that cerebral energy
failure cannot be the cause of the impairment.
Following this line of reasoning, many investigators have now focused
attention on other changes associated with hyperammonemia and hepatic
encephalopathy. One is the phenomenon of astrocyte swelling in
hyperammonemia-induced brain pathologies. Astrocytes contain most of
the brain's activity of the enzyme glutamine synthetase (GSase), which
ostensibly detoxifies ammonia by combining it with glutamate to form
glutamine (20). Thus, during hyperammonemia, glutamine
accumulates in the brain and astrocytes then become swollen and convert
to "Alzheimer type II astrocytes" (21); intracranial
hypertension ensues. Indeed, administration of the GSase inhibitor
methionine sulfoximine in experimental hyperammonemia removes many of
these symptoms (12, 13, 26-28). In particular, the Hirata et al. (12) study concludes that the physical
astrocyte swelling per se may not be the cause of certain symptoms, but that dysfunction of astrocytes related to glutamine accumulation may be key.
In another important recent focus of research, a number of
investigators examined the effects of hyperammonemia on glutamate neurotransmission [reviewed by Butterworth (4)]. This
research reveals that ammonia has complex effects on several classes of glutamate receptors (e.g., AMPA, NMDA, etc.) depending on whether hyperammonemia is acute or chronic. Notably, in acute hyperammonemia, hyperactivation of NMDA receptors appears to lead to cell perturbations via uncontrolled Ca2+ influxes. Blocking of NMDA receptors
with agonists leads to some relief of hyperammonemic symptoms.
In marked contrast to this ammonia sensitivity in mammals, some lower
vertebrates, especially fish, appear to be much more ammonia tolerant,
but the sequelae of events associated with hyperammonemia are much less
well studied (2). For a recent review, see Ref. 14. One extreme piscine example is the gulf
toadfish, Opsanus beta. It can tolerate up to 10 mM [the
96-h half-maximal lethal concentration value (LC50)] of
ammonia in the surrounding water, which rapidly equilibrates across the
gill epithelium and blood-brain barrier (29). Experiments
exposing Opsanus beta to even a sublethal dose of ammonia in
the water (3.5 mM) increased plasma total ammonia concentration from
250 to 1,250 µM, with brain ammonia levels increasing from 1,500 to
4,500 µmol/kg, a value well above the lethal brain ammonia
concentration in mammals cited above. The physiology of these fish
showed some alteration, as indicated by a brief transitory loss of
balance and a longer-term increase in urea excretion compared with
controls, but death did not ensue (29). Given this extreme
ammonia tolerance in the toadfish, the remaining uncertainties of the
mechanisms of ammonia-induced brain neuropathies in mammals, and the
limited treatment options for hyperammonemia in a clinical context, it
seemed worthwhile to pursue the mechanisms of ammonia tolerance in the toadfish.
The present study begins work with the toadfish hyperammonemia model
through an assessment of the differences in the impact of ammonia on
brain metabolism in toadfish vs. rats. Although metabolic effects of
ammonia appear to be less directly related to brain pathology than
astrocyte-mediated glutamine accumulation in mammals, we felt that it
was important to first address this possibility in this new model
system, as well as to confirm these observations in parallel in a
mammalian system. Specifically, through two sets of experiments we test
the hypothesis that there are fundamental differences in ammonia's
effects on brain mitochondrial metabolism between toadfish and rats
that can account for the ability of the toadfish brain to tolerate high
levels of ammonia. The first set of experiments investigates the effect
of ammonia on the level of NADH in brain slices from toadfish and rats.
A second set of experiments investigates the effect of ammonia on the
electron transport chain activity of isolated nonsynaptic mitochondria,
specifically measuring the activity of enzymatic complexes of the
electron transport chain with specific inhibitors (rotenone, antimycin,
and cyanide).
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MATERIALS AND METHODS |
Experimental animals.
Gulf toadfish (Opsanus beta) were collected from Biscayne
Bay, FL, using a roller trawl by commercial fisherman. Before being transferred to the holding tanks at the University of Miami Rosenstiel School of Marine and Atmospheric Science campus, the fish were dipped
in freshwater for 2 min, and then in malachite green-formalin (15 mg/l)
for 2-4 h to prevent the ciliate Cryptocaryon irritans infection. For 2 wk the fish were allowed to recover in 20-liter holding tanks with flow-through, sand-filtered, and
ultraviolet-sterilized seawater from Biscayne Bay before being used for
experiments. The water conditions of the holding tank varied depending
on ambient seawater in the bay: temperature (22-26°C), pH
(7.7-7.9), and salinity (30). Natural photoperiod was
used. Fish were fed with frozen squid twice a week. Male Wistar rats
(250-300 g) from Charles River Laboratories were used for these
experiments. They were kept in cages with dry pellets and water.
Brain extraction and slice preparation.
To obtain the brain, fish were anesthetized with MS-222
(3-aminobenzoic acid ethyl ester, methanesulfonate salt 1 g/l buffered with an equal amount of NaHCO3) dissolved in seawater. The
brain was then perfused with heparinized toadfish saline (50 IU/ml, sodium heparin) for 1 to 2 min to remove the blood. The saline composition (modified from Cortland saline) was as follows (in mM): 171 NaCl, 0.88 MgSO4, 0.46 Na2HPO4,
0.48 KH2PO4, 5 NaHCO3, 11 HEPES, 1 CaCl2, 3 glucose, and 2% BSA, pH 7.4, aerated with 0.25%
CO2, balance O2. To achieve this blood-free
preparation, the heart was exposed and an incision was made between
ventricle and atrium to allow implantation of a cannula (PE-90 tubing), which fed into the ventral aorta and was secured at the conus arteriosus by a silk ligation. After decapitation of the fish, the
brain was extracted from the cranium. The brain was allowed to recover
in superfused cold saline and sectioned cranially to 400-µm thick
with a motorized Vibroslice microtome (Campden Instrument). The slices
were then stored in the artificial saline equilibrated with 0.25%
CO2 balance O2 at 24°C for at least 30 min
before being transferred to the recording chamber.
Rat hippocampus slices were prepared according to a protocol reported
earlier (23). In brief, rats were deeply anesthetized with
pentobarbital sodium (60 mg/kg), cooled, and decapitated. The cranium
was opened and the brain was superfused with cold artificial
cerebrospinal fluid (aCSF). The composition of the aCSF was (in mM) 126 NaCl, 3.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 2 MgSO4, 10 glucose, pH 7.4, equilibrated with 95%
O2 and 5% CO2. The brain was hemisected and
hippocampi were dissected from the cerebral hemispheres. Slices of
400-µm thickness were prepared with a mechanical tissue sectioner,
oxygenated with 95% O2-5% CO2 in aCSF, and
stored at 28°C for at least 1 h before transfer to the recording chamber.
Measurement of the redox state of
NAD+.
The brain slices were placed in an interface recording chamber to
record extracellular evoked potential as described in
Pérez-Pinzón et al. (22). Redox shift of
intramitochondrial nicotinamide adenine dinucleotide (NAD) was measured
by rapid scanning spectrofluorometry as described previously (24,
25). The redox state was indicated by the shift of emission
intensity at 450 nm using 337-nm excitation light induced by a pulsed
nitrogen laser for illumination. The maximal amount of reduced NADH in
the mitochondria was evaluated by the fluorescence emitted during
N2 gassing. The technique takes advantage of the fact that
the reduced pyridine nucleotide (NADH) fluoresces much more intensely
than does the oxidized form (NAD+), such that changes in
fluorescence emission indicate shifts in the reduction/oxidation ratio
of this electron carrier. This technique also takes advantage of
previous studies showing that mitochondrial NADH fluorescence signals
were 10-20 times that of cytoplasmic NADH. This effect is thought
to arise from decreased quenching due to intramitochondrial binding
(3, 7, 15).
For a detailed sampling time course, see Fig.
1. In brief, both toadfish and rat brain
slices were allowed to recover from the trauma caused by sectioning for
at least 30 min until a stable spectrophotometric profile of NADH
fluorescence emission could be established. The experiments for rats
and toadfish brain slices were conducted at 37 and 24°C,
respectively, with 24°C representing a midpoint in the normal thermal
range of the gulf toadfish.
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Fig. 1.
Schematic representation of the experimental protocol for
the measurement of NADH by rapid scanning spectrophotometry. Arrows
indicate time of measurements.
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Rat brain slices were exposed to 1 mM ammonia for 45 min, and the
fluorescence measurements were taken at 5, 15, 30, and 45 min. The
brain slices were then allowed to recover in ammonia-free saline for 30 min before another measurement was made. Finally, the completely
reduced state for NADH was achieved with 15 min of anoxia (nitrogen gas).
The toadfish brain slices were treated with 1 mM ammonia for 60 min
followed by exposure to 6 mM ammonia for 60 min. The fluorescence readings were taken at 15, 30, and 60 min in the respective treatment. With a subsequent 30-min recovery in ammonia-free saline, the fish
brain slices were then brought to anoxic condition induced by a 30-min
exposure to nitrogen gas.
Mitochondria extraction from brain.
All isolation procedures were carried out on ice. The procedure for the
isolation of mitochondria from brain is as described in Lee et al.
(19) with minor changes. The brains from 15 fish were
pooled for an experiment. The fish were anesthetized with MS-222 (0.75 g/l buffered with an equal amount of NaHCO3). The brains
were removed and kept in an isolation buffer containing (in mM) 0.25 sucrose, 10 HEPES, 0.5 EDTA, 0.5 EGTA, pH 7.4, to which BSA (fraction
V, 1 mg/ml) and protease (Nagarse; EC 3.4.21.62, 2.5 mg/g tissue) were
added. It took on average 20 min to remove all 15 brains from the fish
skulls. One rat brain was used for each experiment. Rats were
anesthetized with a mixture of gases: 70% N2O, 26%
O2, and 3-4% halothane. The rat was decapitated
immediately after anesthesia, and the forebrain was kept in the same
isolation buffer as for the fish. For either of the animals, the
brain(s) was (were) chopped and homogenized (8 up and down strokes) in a 15-ml Glas-Col homogenizer (cat. no. 099C S35) with 110- to 150-µm
clearance. This homogenate was centrifuged in a Sorvall RC2-B
centrifuge at 2,000 g for 3 min to separate the membrane constituents from mitochondria and synapses. The resulting supernatant was then centrifuged at 12,000 g for 8 min. The pellets were
resuspended in the isolation buffer with BSA (1 mg/ml) and centrifuged
at 12,000 g for 10 min. The pellets were resuspended in a
0.25 M sucrose solution and centrifuged at 12,000 g for 10 min. The supernatant was discarded, and the pellets were resuspended in
the remaining sucrose solution from the centrifuge tubes. The yield of
mitochondria was 3.66 ± 1.16 mg/ml for the rat brain and
3.43 ± 1.36 mg/ml for fish brains.
Measurements of electron transport chain activity.
All measurements of oxygen consumption were performed at 30°C in a
buffer composed of (in mM) 150 sucrose, 25 Trizma base, 10 potassium
phosphate, pH 7.4. The experiments were divided in two series:
series I tested the oxygen consumption of isolated mitochondria from fish caught during the summer with the administration of 1 mM ammonium chloride in the buffer. Series II tested
the oxygen consumption of isolated mitochondria from fish caught in the
fall with the introduction of 6 mM ammonium chloride in the buffer. The
respiratory and phosphorylating activities of isolated mitochondria
were determined polarographically by using two Clark-type oxygen
electrodes. A Clark-type electrode inserted in a 1.8-ml glass chamber
was used to measure the coupling between the oxidation of substrate
material (pyruvate and malate) and the phosphorylation of ADP for
series I experiments. The same electrode was also used to
measure the oxygen consumption of mitochondria when various enzymatic
complexes of the respiratory electron chain were inhibited in
series I and II. Another type of Clark electrode
with adjustable chamber volume (the OXYGRAPH SYSTEM by Hansatech
Instruments) was used to determine the coupling activity of
mitochondria for series II experiments.
To measure the coupling between the oxidation of substrates and the
phosphorylation of ADP, mitochondria were suspended in a reaction
buffer along with 5.55 mM pyruvate and 2.7 mM malate. ADP (0.16 mM) was
injected at various times to provide substrate for oxidative
phosphorylation. To measure the oxygen consumption of mitochondria
depending on the redox state of enzymatic complexes of the electron
transport chain, mitochondria were introduced in the reaction buffer
along with 5.55 mM pyruvate, 2.7 mM malate, and 0.5 mM ADP
(saturating). Rotenone (3.33 µM dissolved in ethanol) was added to
stop the transfer of electrons from NADH dehydrogenase (complex I) to
ubiquinone-cytochrome c oxidoreductase (complex III).
sn-Glycerol 3-phosphate (G-3-P; 4.17 mM, the substrate for the glycerol-3-phosphate shuttle), which brings electrons directly to
complex III, was added along with 4.44 mM succinate, the substrate for
succinate dehydrogenase (complex II) that passes electrons from complex
II to III. Antimycin (0.055 µM dissolved in ethanol) was added to
block the transfer of electrons from cytochrome b (located within
complex III) to cytochrome c. Ascorbate (0.5 mM) and TMPD
(N,N,N',N'-tetramethyl-p-phenylenediamine,
dihydrochloride; 0.18 mM) were then added. Ascorbate reduces TMPD,
which serves as a substrate for cytochrome oxidase (complex IV). Sodium
cyanide (0.88 mM dissolved in water) was finally used to block
cytochrome oxidase (complex IV), more specifically cytochrome
a3. Appropriate solvent controls were conducted. Protein
estimation on the isolated mitochondria was determined by the
microassay procedure from BioRad protein assay (Coomassie blue).
Statistical analysis.
Data are presented as means ± SE. Paired Student's
t-test was used to compare the significant difference
between paired observations.
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RESULTS |
Brain slices.
In toadfish brain, NADH levels did not significantly change from the
control condition after 60 min of exposure to either 1 or 6 mM ammonia
(Fig. 2A). In contrast, in the
rat brain, NADH levels experienced a significant decrease after even 5 min of exposure to the lower (1 mM) ammonia concentration, stabilizing at a 12% decrease from control values after 45 min. The 30-min recovery period did not reverse the effect of ammonia exposure (Fig.
2B).
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Fig. 2.
A: NADH levels, indicated by the amount of
fluorescence measured at 450 nm, in fish brain slices exposed to 1 and
6 mM ammonia. Open bar, control; light gray bars, 1 mM
NH4Cl; dark gray bars, 6 mM NH4Cl; hatched bar,
recovery; black bar, reduced (N2 gas). A paired 2-tailed
t-test was performed. B: NADH levels, indicated
by the amount of fluorescence measured at 450 nm, in rat brain slices
exposed to 1 mM ammonia. Open bar, control; light gray bars, 1 mM
NH4Cl; hatched bar, recovery; black bar, reduced
(N2 gas). A paired 2-tailed t-test was
performed, * P < 0.02; ** P < 0.01; *** P < 0.001.
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Isolated mitochondria.
The values of phosphorus-to-oxygen ratio (P/O) and respiratory control
index (RCI) (Tables 1 and
2) indicated good viability of the
mitochondrial preparations.
For both toadfish and rat mitochondria, the P/O was not significantly
affected by the addition of ammonium chloride compared with control
values in nearly all cases except for an increase in rat at 1 mM (Table
1). The RCI was also unaffected by the addition of 1 or 6 mM ammonia
for isolated rat brain mitochondria (Table 2). However, the RCI for
toadfish brain mitochondria was significantly lower (by 12%) when
exposed to 1 mM ammonia compared with its control. However, the
addition of 6 mM ammonia in the media did not affect the RCI of
mitochondria isolated from the toadfish compared with control values
(Table 2).
The oxygen consumption rates of isolated mitochondria from both rat and
toadfish brain subjected to 1 or 6 mM of ammonia in the presence of
substrates and inhibitors of the enzymes of the electron transport
chain were not significantly different from the control values (Table
3).
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Table 3.
Rates of oxygen consumption for isolated mitochondria from rat and
toadfish brain subjected to different sets of substates and
inhibitors for experiments in series I and II
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DISCUSSION |
Effects of hyperammonia on NADH levels and rates of oxidative
phosphorylation in rat brain mitochondria.
In the current study, we confirmed that ammonia had a modest effect on
the amount of NADH in rat brain (Fig. 2B) with a 12% decrease in intramitochondrial NADH during ammonia exposure. Previous in vivo studies showed that when rats were subjected to acute ammonia
injection, there was a decrease in the NADH/NAD+ ratio in
the mitochondrial matrix and an increase in NADH/NAD+ ratio
in the cytosol in brain (9, 11, 17). Because our in vitro
experiment was able to replicate in vivo results (where systemic
adaptive responses cannot be ruled out), our observations confirm that
the effects of ammonia on the level of NADH are the direct result of
ammonia acting on brain tissue, rather than via an indirect effect
involving ammonia eliciting a systemic response (i.e., in other
tissues), which subsequently impact the brain. That these effects are
modest in the in vitro situation further confirm the growing
appreciation that hyperammonemia effects on CNS function likely do not
derive largely from impacts on energy metabolism.
It is still instructive to try to explain the factors accounting for
the decrease in the amount of NADH. A significant decrease in NADH in
the mitochondrial matrix could be due to a disruption of the enzymatic
activity of the Krebs cycle, which provides most of the NADH to the
mitochondria, and/or due to an increase in consumption of NADH by
elevating the activity of the complexes of the electron transport
chain, which may or may not be linked to an increased energy demand in
a high-ammonia insult. Considering first the production of NADH, many
studies have shown that the activities of the enzymes of the Krebs
cycle and of the malate-aspartate shuttle in mammalian brain are
affected by ammonia. Albrecht and Faff (1) found that
pyruvate carboxylase, malate-aspartate shuttle enzymes (aspartate
aminotransferase and malate dehydrogenase), and 
-ketoglutarate
dehydrogenase all had decreased activity when the isolated mitochondria
were subjected to ammonia, all potentially contributing to decreased
NADH output. Lai and Cooper (18) demonstrated that brain
-ketoglutarate dehydrogenase complex was directly affected by
ammonia. The study by Kosenko et al. (16) demonstrated that rats given an intraperitonial ammonia injection (and showed convulsion) also had a significant reduction in malate dehydrogenase and succinate dehydrogenase activity, which was previously suggested by
Fitzpatrick et al. (8). The activity of the Krebs cycle and the malate-aspartate shuttle are key elements for the supply of
NADH to the mitochondria, a decrease in the activity of the shuttle
(malate dehydrogenase and aspartate aminotransferase) and a decrease in
-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate
dehydrogenase would reduce the amount of NADH present in the matrix. It
is unlikely that glutamate dehydrogenase could account for the decrease
in NADH via excess glutamate formation in the rat, because it appears
to largely function in the direction of ammonia production in brain,
even during hyperammonemia (5), and glutamate production
would represent a significant drain on important Krebs cycle
intermediates such as 2-oxoglutarate.
To test the second possibility, namely an increase in NADH consumption,
the rates of oxidative phosphorylation of the electron transport chain
enzymes were examined in isolated brain mitochondria. Standard P/O and
RCI measurements showed no major changes with ammonia exposure in
either species (see Tables 1 and 2). The measurements on the rates of
oxygen consumption when manipulating the activity of the different
electron transport chain enzymes also showed no significant variation
with ammonia exposure (see Table 3). Our data clearly indicate that
there is no direct effect of ammonia on the enzymes of the electron
transport chain in rat brain.
Effect of hyperammonia on NADH levels and rates of oxidative
phosphorylation in toadfish brain mitochondria.
Opsanus beta, the gulf toadfish, is a potentially important
animal model for ammonia toxicity studies (29). We
speculated that its ability to resist hyperammonemia at the cellular
level could be due to differences in its brain mitochondrial
physiology. Hyperammonemia in brain slices from toadfish did not affect
NADH levels at the lower concentration employed (1 mM) where rat brain slices were impacted (Fig. 2, A and B). Even 6 mM
ammonia did not significantly affect NADH levels in toadfish brain
slices (Fig. 2A). There are virtually no data available for
the effect of ammonia on enzymes that produce NADH from the toadfish
brain. On the basis of the data of the current study, it would be
interesting to see if these toadfish enzymes have similar or different
ammonia sensitivities to those from rat.
On the basis of our results, it is likely that the exceptional
tolerances of toadfish to high ammonia are likely due to other aspects
besides disruptions of energy metabolism. Because astrocytic glutamine
production appears to be key in mammalian brain susceptibility, the
factors affecting glutamine production and disposal rates in toadfish
brain should be examined in detail. Notably, Wang and Walsh
(29) found rather high activities of GSase in gulf toadfish brain and their multiple fish species. Comparisons within the
family Batrachoididae (toadfish and midshipmen) showed that a species
ammonia LC50 was directly correlated with its brain GSase
activity. This result is at first surprising based on mammalian results, where GSase inhibition ameliorates hyperammonemic effects. However, whereas in mammals brain glutamine concentrations double or
triple under hyperammonemic conditions (27), in gulf
toadfish, subjected to conditions that increase brain ammonia levels by several fold (i.e., well above lethal levels in mammals), total brain
glutamine content only rose by 30% (29). These
observations suggest that in contrast to mammals, during hyperammonemia
in toadfish, brain glutamine dynamics may differ in rate of production by astrocytes and/or rate of glutamine clearance from the brain and
plasma. Clearly, to begin to test this hypothesis, brain glutamine compartmentation and turnover will need to be examined under a carefully graded series of hyperammonemic exposures. An equally inviting possibility to pursue in parallel would be to examine brain
fluid content and intracranial pressure under these circumstances to
see if the increases seen in mammals even occur in toadfish. Such
studies would extend the potential usefulness of the toadfish as a
model system for studies of hyperammonemia and hepatic encephalopathy.
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ACKNOWLEDGEMENTS |
We are grateful to Dr. G. Xu for advice on experimental manipulations.
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FOOTNOTES |
These studies were supported by National Institutes of Health
Grants NS-05820 to M. A. Pérez-Pinzón and ES-11005 to
P. J. Walsh, and by a pilot project grant from the
University of Miami NIEHS Marine and Freshwater Biomedical Science
Center (ES 05705).
Address for reprint requests and other correspondence: C. Veauvy, RSMAS/MBF, Univ. of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149 (E-mail: cveauvy{at}rsmas.miami.edu).
1
Ammonia can exist as either an ion
(NH4+) or a dissolved gas (NH3),
and the 2 species are interconverted by the rapid and nonenzymatic
removal/addition of a proton. The pKa of this reaction is ~9, so that
at physiological pHs (~7), the ratio of ionic/nonionic ammonia is
~100. When we refer to ammonia in this paper, we mean total ammonia
unless a particular chemical form is specified.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 30, 2002;10.1152/ajpregu.00018.2002
Received 11 January 2002; accepted in final form 22 May 2002.
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