Effects of thiopental anesthesia on local rates of
cerebral protein synthesis in rats
C. Beebe
Smith,
C.
Eintrei,
J.
Kang, and
Y.
Sun
Laboratory of Cerebral Metabolism, National Institute of Mental
Health, United States Public Health Service, Department of Health
and Human Services, Bethesda, Maryland 20892
 |
ABSTRACT |
We have examined the
effects of a surgical level of thiopental anesthesia in adult male rats
on local rates of cerebral protein synthesis with the quantitative
autoradiographic
L-[1-14C]leucine
method. The relative contribution of leucine derived from protein
breakdown to the intracellular precursor amino acid pool for protein
synthesis was found to be statistically significantly decreased in the
anesthetized rats compared with controls. In the brain as a whole and
in 30 of the 35 brain regions examined, rates of protein synthesis were
decreased (1-11%) in the anesthetized rats. Decreases were
statistically significant (P
0.05)
in the brain as a whole and in six of the regions, and they approached statistical significance in an additional 13 regions, indicating a
tendency for a generalized but small effect.
leucine; brain; amino acid recycling; barbiturate
 |
INTRODUCTION |
PROTEIN METABOLISM IN BRAIN is essential for both
maintenance and growth of tissue, and it may underlie some of the
special functions of the nervous system such as plasticity and memory formation. The effects of various pharmacological agents on brain protein synthesis are largely unknown. It is important to appreciate such effects because they may confound results of animal experiments or
produce side effects in patients. Anesthetics, which are widely used in
animal research in neuroscience, have been reported to cause large and
generalized decreases in rates of protein synthesis in brain (4, 5, 8).
These previous studies were beset with methodological problems. We have
therefore reexamined the question of effects of barbiturate anesthesia
on regional rates of protein synthesis in brain. In the present study,
rates of protein synthesis were determined with a quantitative
autoradiographic tracer method that measures the rate of incorporation
of
L-[1-14C]leucine
into protein (17). The method takes into account the dilution of the
specific activity of the precursor amino acid pool in the tissue by
unlabeled leucine derived from protein degradation. The study was
designed to measure the effects of a surgical level of thiopental
anesthesia on the rates of protein synthesis in localized regions of
brain and in the brain as a whole, weighted for the masses of its
component parts. Our results indicate that, in the brain as a whole and
in most of the 35 brain regions examined, rates of protein synthesis
are slightly decreased (5-11%) in thiopental-anesthetized rats.
 |
METHODS |
Chemicals.
Chemicals and materials were obtained from the following sources:
L-[1-14C]leucine
(sp act 55 mCi/mmol) from NEN, Wilmington, DE;
L-[4,5-3H]leucine
(sp act 180 Ci/mmol) from American Radiolabeled Chemicals, St. Louis,
MO; Escherichia coli tRNA from Sigma,
St. Louis, MO; vanadyl ribonucleoside complex and redistilled nucleic
acid-grade phenol from Bethesda Research Laboratories, Gaithersburg,
MD; L-norleucine from
Cyclochemicals, Division of Travenol Laboratories, Los Angeles, CA; and
5-sulfosalicylic acid from Fluka Chemie, Buchs, Switzerland.
Animals.
All procedures were carried out in accordance with the National
Institutes of Health Guidelines on the Care and Use of Animals and an
animal study protocol approved by the National Institute of Mental
Health Animal Care and Use Committee. Thirty-two normal male
Sprague-Dawley rats (Taconic Farms, Germantown, NY) weighing 243-320 g were studied. Food and water were provided ad libitum. Rats were maintained under controlled conditions of normal humidity and
temperature with standard alternating 12-h periods of light and
darkness. Rats were prepared for experiments by insertion under light
halothane anesthesia of polyethylene catheters into one femoral artery
and both femoral veins. Catheters were tunneled under the skin to exit
at the nape of the neck so that the rats could not gain access to the
tubing. At least 3 h were allowed for recovery from the 20-min surgery
before initiation of the experimental procedure. The experimental
animals were reanesthetized with intravenous thiopental sodium
(40-60 mg/kg) to a surgical level as indicated by the extinction
of the corneal reflex (1, 16). Rats were maintained throughout the
experimental procedure at this level by intravenous administration of
thiopental sodium as required. Rats were anesthetized for 60 min before
the initiation of measurements. Control animals were allowed to remain
conscious and freely moving.
Physiological variables.
Physiological variables were measured to evaluate each animal's
physiological state. Mean arterial blood pressure, pH,
PCO2 and
PO2, hematocrit, arterial plasma
glucose concentration, and rectal temperature were measured as
previously described (21).
Procedure to determine
WB,
WB, and
i.
i is the ratio of the
integrated specific activity of leucine in the tissue precursor pool to
that of the arterial plasma during at least 60 min after an intravenous
pulse of labeled leucine
|
(1)
|
where
and
Cpp are the concentrations of the
labeled and unlabeled leucine, respectively, in the precursor pool in tissue i, and
and
Cp represent the concentrations in
arterial plasma of labeled and unlabeled leucine, respectively. If the
(t)/Cp
is held constant for a long enough time (
) for the tissue free and
tRNA-bound leucine pools to reach a steady state with the plasma, then
|
(2)
|
The
time necessary to achieve this apparent steady state for leucine in the
normal, conscious rat is between 30 and 60 min (17). The steady state
is designated as apparent because it pertains to the free and
tRNA-bound leucine pools only; a steady state for the
[3H]leucine
incorporated into peptide bonds in the protein is not even approached
during the 60- or 90-min experiments.
Analogously,
i is the
steady-state ratio of the specific activity of leucine in the tissue
acid-soluble amino acid pools to that of the arterial plasma
|
(3)
|
where
and
CE are the concentrations in the
extracellular space of labeled and unlabeled leucine, respectively, and
and
CM are the concentrations in the
intracellular metabolic pool of labeled and unlabeled leucine,
respectively.
WB and
WB were evaluated in 12 rats by
the method described by Smith et al. (17). Briefly, a constant arterial
plasma specific activity for
[3H]leucine was
maintained for 60-90 min by means of a programmed infusion of
[3H]leucine. The
specific activities of
[3H]leucine in
arterial plasma and in the acid-soluble and tRNA-bound pools in brain
were determined as described below.
Extraction and purification of aminoacyl-tRNA.
The brain from each rat was homogenized by a motor-driven loose-fitting
all-glass homogenizer in 10 ml of 0.25 M sucrose (0°C) containing
10 mM vanadyl ribonucleoside complex to inhibit RNase, 6 mg of
uncharged E. coli tRNA as carrier, and
L-norleucine (0.02 mM) added as
an internal standard. The homogenates were centrifuged at 100,000 g for 1 h, and a pure aminoacyl-tRNA
fraction was isolated from the supernatant fraction, as previously
described (18). Briefly, the supernatant fraction was treated with TCA,
the precipitated protein and nucleic acid were washed repeatedly to
remove free amino acids, and the aminoacyl-tRNA was separated from
protein by phenol extraction. The aminoacyl-tRNA was hydrolyzed at pH 10, and the specific activity of the previously tRNA-bound but now free
amino acids was determined as described below.
Extraction of acid-soluble fraction in brain tissue.
A 100-µl volume of the cytosol fraction, i.e., the supernatant
solution derived from the 100,000 g
centrifugation of the whole brain homogenates, was deproteinized by the
addition of an equal volume of a solution of 8% (wt/vol)
sulfosalicylic acid and stored at
70°C until assayed for
leucine and
[3H]leucine
concentrations. At the time of these assays, the samples were thawed,
mixed, and centrifuged for 30 min at 5,000 g at 4°C to remove the
precipitated protein.
Dissection of brain regions.
Brains were placed dorsal side up in a stainless steel rodent brain
matrix (Activational System, Warren, MI), which was kept cold on
crushed ice, and blades (Thomas Scientific, Swedesboro, NJ) were
inserted through the slots in the matrix at right angles to the
sagittal axis down to the level of cerebellum. The brains were sliced
at intervals of 1 mm. Brain regions were dissected from these coronal
slices (6). The remainder of the brain was placed on the chilled glass
plate, and the cerebellum was completely detached from the brain stem
and set aside. The hypoglossal nuclei were dissected bilaterally from
the brain stem (24). Dissected brain regions were weighed, homogenized
in 4% sulfosalicylic acid, which contained 2.5 µM norleucine as an
internal standard, and stored at
20°C until assayed for
leucine and
[3H]leucine
concentrations. At the time of these assays, the samples were thawed,
vortexed, and centrifuged for 30 min at 5,000 g at 4°C to remove the
precipitated protein.
Assay of specific activity of
[3H]leucine.
Specific activities of
[3H]leucine in
deproteinized plasma, tissue acid-soluble fractions, and fractions
derived from the deacylation of the aminoacyl-tRNA were assayed by
post-column derivatization with
o-phthaldehyde and fluorometric assay
with a Beckman amino acid analyzer (model 7300, Beckman Instrument,
Fullerton, CA). This system can measure 10-100 pmol of leucine
with a 3% coefficient of variation. Fractions, after passage through
the detector, were collected every minute and assayed for
3H with a Tri-Carb liquid
scintillation analyzer (model 2250CA, Packard Instrument, Downers
Grove, IL). Specific activity was calculated from total
3H in all fractions in the leucine
peak and the total measured leucine content in the peak. The leucine
concentration and the specific activity of
[3H]leucine in the
acid-soluble pool in the tissue were corrected for contamination by the
leucine in the blood contained in the tissue. The equilibrium
distribution of free leucine between erythrocytes and plasma was
measured and found to be 0.67, and the hematocrit in brain was
determined to be 30% (22).
Calculation of values of
WB and
WB.
Values of
WB were calculated
from the ratio of the measured steady-state specific activity of the
tRNA-bound leucine in the tissue to that of the acid-soluble leucine in
arterial plasma (Eq. 2), and values
of
WB were calculated from the
ratio of the measured steady-state leucine specific activity of the
brain acid-soluble pool to that of the arterial plasma
(Eq. 3). The time course of the
specific activity in arterial plasma and the specific activities of
acid-soluble and tRNA-bound leucine in whole brain at the end of the
experimental period were determined as described above. The apparent
steady-state free leucine specific activity in the arterial plasma was
calculated as the mean of the specific activities determined from 40 min to the end of the experimental period. Whereas in some of the
experiments the specific activity of leucine in the arterial plasma is
not constant during the first 30 min, from 40 to 60 min (60-min
experiments) and from 50 to 90 min (90-min experiments), values were
within ±10% of the mean and showed no overall trend to increase or
decrease over the entire interval. Leucine from the tRNA-bound amino
acid fractions was uncontaminated by leucine derived from any blood in
the brain tissue because of the procedure used to separate this
fraction from the free amino acids in the tissue. The specific activity
of [3H]leucine in the
acid-soluble pools in the tissue was corrected for leucine in the
residual blood contained in the tissue as described above.
Determination of local rates of protein synthesis.
Local rates of protein synthesis were determined in 10 control and 10 thiopental-anesthetized rats. Rats were surgically prepared and
catheterized, and their physiological states were monitored as
described above. The procedure for the determination of the local rate
of cerebral protein synthesis
(lCPSLeu) has been previously described (22). Briefly, the administration of an intravenous pulse of
L-[1-14C]leucine
(100 µCi/kg) initiated the procedure. Timed arterial blood samples
were collected during the following 60 min for determination of the
time courses of plasma concentrations of leucine and
[14C]leucine. At the
end of the 60-min experimental period, rats were killed by an
intravenous injection of pentobarbital sodium, and the brains were
rapidly removed and frozen in isopentane cooled to
40°C with
dry ice. Brain sections were prepared and autoradiographed along with
calibrated
[14C]methylmethacrylate
standards as previously described (22). The rates of leucine
incorporation into protein in individual brain regions and the average
for the brain as a whole, weighted for the relative masses of its
component parts, were determined by analysis of the autoradiograms with
a computerized image-processing system (MCID Imaging Research, St.
Catharines, ON, Canada) with a pixel size of 28 µm. Regions were
located according to the rat brain atlas of Paxinos and Watson (13).
The concentration of 14C in each
region of interest in the autoradiograms was determined from the
optical density vs. 14C
concentration curve for the calibrated plastic standards. Local rates
of protein synthesis were calculated by means of the operational equation of the method (17) with the condition-specific values of
i
|
(4)
|
where
Ri is the rate of leucine
incorporation into protein in tissue
i;
(T)
is the concentration of 14C fixed
in the tissue i at any time,
T, after introduction of the tracer
into the circulation;
i is
equal to the fraction of leucine in the precursor pool for protein
synthesis in the tissue i that is
derived from plasma; and t is variable
time. Rates of protein synthesis measured with the
[14C]leucine method
are rates of incorporation of leucine into all tissue protein, which is
comprised of a mixture of many individual proteins. The rate of leucine
incorporation into each individual protein is weighted according not
only to its individual rate of turnover but also to the fraction of
total tissue protein that it comprises.
Statistics.
Values of
WB,
WB,
i, and
i and weighted average rates
of protein synthesis and lCPSLeu
in 35 brain structures determined in thiopental-anesthetized rats were
compared with those of controls by means of Student's
t-tests. Results from one control
animal were not included in the series because its endogenous arterial
plasma leucine concentration decreased by 32% during the course of the
60-min experiment.
 |
RESULTS |
Physiological status.
Sixty minutes of surgical anesthesia with thiopental produced
statistically significant changes in some of the physiological variables that were measured (Table 1).
Arterial blood PO2 was decreased
16%, PCO2 was increased 28%, and pH
was decreased 1% in the anesthetized rats compared with the controls. Arterial plasma glucose concentration was 17% lower and mean arterial blood pressure was 9% lower in the anesthetized rats. Rectal
temperature and hematocrit were unchanged. The behavior of the control
animals was normal; they were alert but calm. The anesthetized animals were unresponsive to stimulation throughout the experimental period.
Effects of thiopental anesthesia on leucine concentrations in brain
amino acid pools.
Leucine concentrations in both the total acid-soluble and tRNA-bound
amino acid pools extracted from whole brain of the anesthestized rats
were statistically significantly higher than those of controls (Table
2). Tissue acid-soluble to plasma
distribution ratios, however, were similar in the two groups,
indicating that the higher leucine concentration in the acid-soluble
pool merely reflects a higher arterial plasma leucine concentration in
the anesthetized animals.
Effects of thiopental anesthesia on values of
i and
i.
In 12 thiopental-anesthetized rats, a constant arterial plasma
[3H]leucine
concentration was maintained for a duration of 60 (n = 4) or 90 (n = 8) min, and the ratios of
[3H]leucine specific
activities in tissue pools to that of arterial plasma were determined
at the end of the infusion. The ratio in the tRNA-bound pool was
similar at both time points, 0.62 ± 0.01 at 60 min and 0.64 ± 0.01 (mean ± SE) at 90 min, indicating that a steady state had been
achieved by 60 min. In the acid-soluble pool, the ratio increased
between 60 and 90 min from 0.52 ± 0.01 to 0.59 ± 0.01 (mean ± SE); these values are statistically significantly different from
one another (P = 0.0006, Student's
t-test), indicating that the
acid-soluble pool turns over more slowly and has a longer half-life
than the tRNA-bound pool. We have assumed that a steady state was
achieved in the acid-soluble pool by 90 min. In conscious rats, the
tRNA-bound and acid-soluble pools reach apparent steady states after 30 and 60 min, respectively, of a constant arterial [3H]leucine
concentration. It is not unreasonable to allow 30 min beyond the
attainment of a steady state in the tRNA-bound pool for the
acid-soluble pool to reach a steady state with the plasma in the
thiopental-anesthetized rats. We have therefore used the ratios at 90 min of 0.64 and 0.59 as the values for
WB and
WB, respectively. Both of these
values are statistically significantly (P < 0.001) higher than the values
of
WB and
WB in conscious adult rats
(22).
We have previously shown that in conscious adult rats, values of
WB correlate closely with the
values of
WB (22) and that the
relationship could be fitted quite well by a linear equation (
WB = 0.29 + 0.60
WB). The best-fitting
linear relationship between
WB
and
WB is unchanged by the
addition of the results of our 90-min experiments in the
thiopental-anesthetized rats to the original series of controls (Fig.
1). The correlation between
WB and
WB for the combined group
remains statistically significant (rxy = 0.91, P < 10
6), and agreement
between the measured values of
WB and those estimated by the
linear equation was excellent. No statistically significant improvement
in fit was found with polynomial regressions of higher degrees.

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Fig. 1.
Relationship between experimentally determined values of ratios
WB and
WB. Each point represents
determinations of both values in a single rat. Ratios were determined
in 9 conscious rats ( ) and 8 thiopental-anesthetized rats ( ).
Best-fitting straight line and equation for line are illustrated.
Correlation coefficient for fit was 0.91 (P < 10 6).
|
|
Regional values of
i were
estimated from the linear relationship between
WB and
WB and experimentally
determined values of
i. Four
thiopental-anesthetized rats already in a steady state for unlabeled
leucine were given programmed intravenous infusions of
[3H]leucine that
maintained the specific activity of
[3H]leucine in
arterial plasma relatively constant long enough (90 min) for the
acid-soluble leucine pool in brain tissue to reach an approximate
steady state with respect to plasma. In all four experiments, the
specific activities of the acid-soluble leucine pool in all 13 regions
examined were below those of arterial plasma. The values of
i ranged from 0.45 in
substantia nigra to 0.60 in the hypoglossal nucleus (Fig.
2). The data were statistically analyzed
for homogeneous subgroups of these values by repeated-measures ANOVA.
One subgroup was found within which there were no significant differences in the values of
i; this group was composed of
all regions of gray matter except substantia nigra, globus pallidus,
and hypoglossal nucleus. The mean ± SE value in this group was 0.54 ± 0.007. The values in the other regions were outside the range of
this homogeneous subgroup. Values for
i (Table
3) were calculated for each region based on
the linear equation (Fig. 1) fitted from the determined values of
WB and
WB. Examination of these data
for homogeneous subgroups by repeated-measures ANOVA showed the same
subgroup of gray matter as was found with the values of
i.

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Fig. 2.
Experimentally determined regional values of
i in thiopental-anesthetized
rats. Data are mean ± SE (bars) values from determinations in 4 rats in all regions except globus pallidus and internal capsule, in
which determinations were made in 3 rats. Regional values were tested
for homogeneity by means of repeated-measures ANOVA. One subgroup of
gray matter regions (see RESULTS)
was found to be homogeneous. Abbreviations are as follows: SN,
substantia nigra; GP, globus pallidus; V CX, visual cortex; A CX,
auditory cortex; Thal, thalamus; MG, medial geniculate; IColl, inferior
colliculus; F CX, frontal cortex; Acb, nucleus accumbens; CP, caudate
putamen; IC, internal capsule; CC, corpus callosum; XII, hypoglossal
nucleus.
|
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Effects of thiopental anesthesia on
lCPSLeu.
Autoradiograms from anesthetized rats were not apparently different
from those of controls (Fig. 3). Rates of
leucine incorporation into protein were determined in the brain as a
whole and in 35 brain regions (Table 4). In
the brain as a whole, lCPSLeu was decreased by 9% (P = 0.026) in the
anesthetized rats. In 30 of the 35 regions examined, rates of protein
synthesis were lower in the anesthetized rats than in the controls.
Decreases in lCPSLeu were
statistically significant (P
0.05)
in six of the regions; the magnitude of these decreases was 9-11%
(Table 4).

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Fig. 3.
Representative
[14C]leucine
autoradiograms from control (left)
and thiopental-anesthetized (right)
rats. Abbreviations are as follows: cc, corpus callosum; CP, caudate
putamen; PC, piriform cortex; ox, optic chiasm; PVN, paraventricular
hypothalamic nucleus; BLA, basolateral amygdaloid nucleus; SC, superior
colliculus; Te3, auditory cortex; MG, medial geniculate nucleus; OC1,
visual cortex; IC, inferior colliculus; Pr5, trigeminal nucleus.
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Table 4.
Effects of thiopental anesthesia on whole brain and regional rates of
leucine incorporation into protein
(nmol · g 1 · min 1)
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|
 |
DISCUSSION |
The results of the present studies show that surgical barbiturate
anesthesia decreases rates of protein synthesis in the brain. The
effects are fairly small in magnitude, i.e., ~10%, and appear to be
generalized. Rates of protein synthesis reported in these studies are
the actual rates of leucine incorporation into protein because the
equation (Eq. 4) used to calculate
lCPSLeu includes a factor,
i, which corrects the
integrated specific activity of the labeled leucine measured in
arterial plasma for the contribution of unlabeled leucine derived from
protein degradation in the tissue. The values of
i used in the calculations were condition specific (conscious vs. thiopental anesthetized) as well
as region specific. Values for
WB were determined
experimentally in control and thiopental-anesthetized rats, and values
for
i were estimated from
measured values of
i for the
specific regions analyzed under both conditions. Without correction for recycling, rates of protein synthesis determined with a radiolabeled amino acid tracer may be underestimated.
Methodological considerations.
Results of previous studies with
[35S]methionine as the
tracer have suggested that large and generalized decreases in rates of
protein synthesis in brain occur with various anesthetic agents (5, 8).
In neither of these studies, however, was dilution of the precursor
pool by unlabeled methionine derived from protein degradation measured.
In addition, there are problems with side reactions and significant
levels of nonspecific labeled metabolic products of
[35S]methionine, which
appear in both the brain and the blood. In both of these studies,
concerns were raised that the effects on protein synthesis may actually
have been due to reductions in body temperature that accompany
anesthesia. In our studies, all of these potential problems have been
circumvented.
In our calculations of lCPSLeu, we
included a factor
i in the
equation to correct for the dilution of the precursor pool by unlabeled
leucine from protein breakdown. We have determined
WB and estimated regional
values for
i under both
control and anesthetized conditions. Values of
i are functions of
steady-state measurements of
i. In control animals, we
verified that a steady state was reached at the time of the measurements, but in the anesthetized rats, we have assumed that a
steady state was reached after 90 min of a programmed infusion of
labeled leucine. If, in some brain regions, a steady state was not
achieved at 90 min, we may have underestimated
i and consequently the true
value of
i. An underestimation of
i would result in an
overestimation of lCPSLeu. The effect on the final results would be an underestimation of the magnitude of the reduction in
lCPSLeu by thiopental.
Extraneous labeled metabolites were avoided by the use of
carboxyl-labeled leucine because the label is transiently converted to
-[1-14C]ketoisocaproic
acid and then to
14CO2.
Labeled CO2 in the plasma is
removed during the deproteinization step, and we have measured the time
course of the appearance of
-[1-14C]ketoisocaproic
acid in the plasma after a pulse injection of [1-14C]leucine in both
controls and thiopental-anesthetized rats. In both cases, the
integrated activity of
-[14C]ketoisocaproic
acid is only 5% of the total integrated activity of the acid-soluble
pool of 14C in the plasma.
Finally, in our experiments, rectal temperature was maintained at
38°C with a heat lamp thermostatically controlled by a thermistor
temperature-sensing probe.
Tissue energy use and protein synthesis rates.
Studies in tissues other than brain have also shown effects of
anesthesia on leucine and phenylalanine metabolism. Studies of whole
body leucine metabolism in dogs (7) have indicated that leucine
incorporation into protein decreases during halothane anesthesia.
Others have reported that, in both perfused lung and perfused heart,
pentobarbital (342 g/ml) and halothane (1-4%) inhibit
phenylalanine incorporation into protein (14). These effects of
pentobarbital on heart were accompanied by 50% decreases in creatine
phosphate; levels of ATP were unchanged. In barbiturate-anesthetized brain, where energy use is reduced (9, 19, 20, 23), high-energy phosphate levels remain normal or are elevated (10, 12), but rates of
protein synthesis appear to be slightly reduced. We have seen changes
in rates of protein synthesis under conditions in which there may be a
depletion of ATP, such as during focal motor seizures (3). During
seizures, rates of glucose utilization were increased severalfold in
the focus and in structures in direct communication with the focus, but
in these same areas, rates of protein synthesis were reduced.
Effects of thiopental anesthesia on values of
i.
Values of
i calculated for
specific brain regions were homogeneous in most gray matter regions
excluding globus pallidus, substantia nigra, and hypoglossal nucleus.
Mean values for the two white matter regions examined were very
similar, but there are not adequate data to confirm that values in all
white matter regions are the same. Values for
i across regions in control
(22) and thiopental-anesthetized rats were compared by
repeated-measures ANOVA. The two groups are statistically significantly different from one another. Values for
i are clearly higher in all
regions in the thiopental-anesthetized rats compared with controls.
Whereas values for
i were
lowest in the globus pallidus and substantia nigra and highest in the
hypoglossal nucleus in both groups, values for
i in white matter were
relatively lower in control animals compared with the anesthetized rats. In the thiopental-anesthetized rats, values in white matter were
among the highest measured, suggesting that changes in rates of either
protein degradation or delivery of amino acids from the plasma are
greater in white matter than in gray matter regions, which may reflect
the high solubility of thiopental in lipids with a consequent higher
tissue concentration in white matter. Values of
WB determined in
thiopental-anesthetized rats were also higher than in controls, but by
20%, indicating that the total tissue acid-soluble leucine pool is
more affected by thiopental than the tRNA-bound pool.
Effects of thiopental anesthesia on leucine transport and protein
degradation.
The results of several studies (2, 15) suggest that barbiturates may
increase transport of neutral amino acids into brain. If so, then the
balance between the contributions to the precursor amino acid pool from
the plasma and protein breakdown could be altered, which would in turn
have an effect on the value of
as follows
|
(5)
|
By
rearranging and substituting
lCPSLeu for the sum of the fluxes
from plasma and protein breakdown
|
(6)
|
and
|
(7)
|
The
flux of leucine from the plasma into the precursor amino acid pool for
protein synthesis calculated from measured rates of protein synthesis
and values of
WB is 3.1 nmol · g
1 · min
1
in both controls and anesthetized rats, indicating no effect on net
transport across the blood-brain barrier. In contrast, the calculated
flux from protein breakdown is decreased by 22% in anesthetized rats
from 2.3 in controls to 1.8 nmol · g
1 · min
1,
indicating a decreased rate of protein degradation in the brain as a
whole under these conditions. In the 13 individual brain regions in
which
i was determined and
i was calculated, results were
similar to those in whole brain; i.e., calculated fluxes of leucine
into tissue precursor pools from plasma were similar under both
conditions, whereas fluxes from protein degradation were consistently
lower in the thiopental-anesthetized rats. The effect of greatest
magnitude (30% decrease) was found in corpus callosum.
Effects of thiopental anesthesia on
lCPSLeu.
Thiopental anesthesia reduced
lCPSLeu in the brain as a whole by
9%. In an effort to determine whether this was a general or regionally
specific effect, we analyzed
lCPSLeu in 35 brain regions. By
standard statistical analysis (Student's
t-tests), six of the regions had
statistically significant decreases in lCPSLeu
(P
0.05). If we apply the very
conservative correction for multiple comparison statistics by the
method of Bonferroni, none of the regions is statistically
significantly affected by thiopental, but the preponderance of
P values (~50%) at or close to
statistical significance (P
0.1)
supports a more generalized effect.
Perspectives.
These investigations show that surgical thiopental anesthesia in rats
results in widespread but small decreases in rates of cerebral protein
synthesis. The results of our analyses of fluxes from protein
degradation suggest that rates of protein degradation may also be
decreased throughout the brain. The biological or clinical significance
of these relatively small effects of thiopental anesthesia is
uncertain. A small inhibition in protein synthesis in the nervous
system counterbalanced by a decrease in breakdown indicates that
turnover is slowed but that there should be no net loss of protein.
Perhaps under anesthesia, when the brain is relatively inactive, a
reduced protein turnover rate would not be harmful to the tissue. The
mechanism by which thiopental effects this reduction is probably not
related to activity per se, since we have found that protein synthesis
rates in adult monkeys are positively correlated with the time in the
inactive state of deep sleep (11). Effects might be larger after more prolonged anesthesia and/or at deeper levels of anesthesia. We cannot ascertain from these studies whether the cells affected are
neurons or glia or both. Interestingly, results for the regions that
are rich in neuronal cell bodies suggest that there is very little
effect on neurons. In contrast, both protein synthesis rates and fluxes
from protein degradation in areas rich in neuropil and white matter
appear to be consistently reduced in the thiopental-treated rats. This
apparent predilection for effects in white matter and neuropil may
reflect the higher solubility of thiopental in these more lipid-rich
areas of brain.
 |
ACKNOWLEDGEMENTS |
We thank Gladys Deibler and Tom Burlin for carrying out the amino
acid analyses, Jane Jehle for preparing tissue sections for
autoradiography, Dr. Karen Pettigrew for expert assistance with the
statistical treatment of the data, and Dr. Louis Sokoloff for helpful
suggestions in preparing the manuscript.
 |
FOOTNOTES |
Address for reprint requests: C. B. Smith, Laboratory of Cerebral
Metabolism, National Institute of Mental Health, Bldg. 36, Rm. 1A07, 36 Convent Dr., MSC 4030, Bethesda, MD 20892-4030.
Received 24 October 1997; accepted in final form 23 January 1998.
 |
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