Department of Physiology and Cellular Biophysics, College of
Physicians and Surgeons, Columbia University, New York, New York
10032
Rat aortic endothelium is differentiated
regionally for three signal pathways capable of regulating the cGMP
content of the underlying smooth muscle. Formation of nitric oxide (NO)
from L-arginine and of glutamate from L-leucine
increase cGMP; however, formation of prostaglandin H2
(PGH2) decreases cGMP. All three have peak activity in the
windkessel area just distal to the aortic arch and decrease
peripherally. We report evidence that the biochemical route of the
leucine-to-glutamate (Leu
Glu) pathway is via metabolism of leucine
to acetyl CoA, that the controlling reaction of the pathway is mediated
by the branched chain
-ketoacid dehydrogenase complex (BCDC), and
that glutamate formation via the Leu
Glu pathway is a major source of
aortic segment free glutamate in vitro. Interruption of the pathway by
treatment of precontracted rat aortic rings in vitro with each of three
classes of inhibitors (leucine analogs, competitors for the BCDC
reaction, or inhibitors of L-glutamate transport) enhances
contractile responses. The enhancement requires an intact endothelium
and is not owing to reductions in NO formation. The results support the
hypothesis that the Leu
Glu pathway functions in the regulation of
aortic contractility and compliance.
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INTRODUCTION |
STUDIES OF RAT AORTIC
SEGMENTS in vitro (1, 40, 41) have demonstrated a
characteristic regional pattern of functional differentiation of the
endothelium for the generation of three classes of signals capable of
regulating the cGMP content of the underlying vascular smooth muscle.
Endothelial formation of nitric oxide (NO) from L-arginine
(1) and of glutamate from L-leucine (41) increase cGMP, which is reported to mediate
relaxation of vascular smooth muscle (21, 35, 38).
Endothelial formation of prostaglandin H2
(PGH2) via the cyclooxygenase arm of the eicosanoid pathway
decreases cGMP (40). All three pathways exhibit a similar regional pattern of activity: maximal values just distal to the aortic
arch, i.e., in the "windkessel region," and progressively decreasing values distally. On the basis of these observations and the
functional importance of the windkessel elastic reservoir, whose
compliance and other viscoelastic properties influence hemodynamic parameters, such as the arterial systolic and pulse pressures, work of
the left ventricle, and pulse-wave velocity (15), we have
hypothesized (41) that an interplay of the NO,
leucine-to-glutamate (Leu
Glu), and PGH2 signal pathways
mediates the dynamic regulation of smooth muscle contractility and
aortic compliance in the windkessel region.
The Leu
Glu pathway, the most recently described of these mechanisms,
is composed of the endothelial uptake of
L-[U-14C]leucine, the relatively efficient
utilization of the leucine carbons for synthesis of
[14C]glutamate, and the transfer of the glutamate to the
underlying smooth muscle via a compartmentalized mechanism
(41). This study characterizes the metabolic route of the
Leu
Glu pathway and examines its role in the regulation of aortic
contractile responses. It is well established that leucine, an
essential, "ketogenic" amino acid, is metabolized via
3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) to acetyl CoA
and acetoacetate by a reaction sequence localized largely in the
mitochondria (12, 47). Acetyl CoA so derived can
enter the tricarboxylic acid cycle by the condensation reaction
with oxaloacetate and result in the formation of
-ketoglutarate, which can be converted to glutamate by the glutamate dehydrogenase reaction or by transamination. The studies below show that the aortic
Leu
Glu conversion utilizes this route. Aortic segments incubated in
vitro with radioactive leucine labeled at different positions or with
other radioactive precursors yield radioactive glutamate and
CO2 as predicted qualitatively and quantitatively by the
catabolic sequence. Thus [U-14C]leucine, which is
ketogenic, yields [14C]glutamate efficiently;
[U-14C]isoleucine, only partially ketogenic, yields
considerably less labeled glutamate; and [U-14C]valine,
not ketogenic, yields insignificant labeled glutamate. Furthermore, the
experimental results indicate that the tissue specificity and overall
activity of the Leu
Glu pathway is determined by three initial
reactions of the catabolic sequence: cellular uptake of leucine,
transamination to
-ketoisocaproate, and conversion of the latter to
isovaleryl CoA via oxidative decarboxylation by the branched-chain
dehydrogenase complex (BCDC). The BCDC reaction is the controlling
reaction whose activity accounts for the regional Leu
Glu pattern of
the rat aortic endothelium.
To examine the role of the Leu
Glu pathway in the regulation of
aortic smooth muscle contractility, we studied the effects of blocking
the pathway on the contractile responses of aortic segments in vitro.
Given prior evidence that Leu
Glu blockade owing to treatment with
leucine analogs decreases the smooth muscle cGMP content
(41), we predicted increased contractile responses. In the
present study, three different classes of compounds capable of
interrupting the Leu
Glu pathway were examined to ensure the specificity of the inhibition: 1) the leucine
analogues L-leucinol and L-leucinamide,
previously shown to block the conversion of [U-14C]leucine to [14C]glutamate and to
decrease aortic segment cGMP (41); 2)
-ketoisovalerate and its amino acid precursor L-valine,
which do not yield acetyl CoA or glutamate but are metabolized by the
same BCDC (36) that functions in the controlling reaction
of the Leu
Glu pathway; and 3) D-aspartate, a
relatively specific and metabolically inert inhibitor of
Na+-dependent glutamate transporters (17, 28,
42). The results demonstrate that interruption of the pathway by
all three classes of compounds significantly enhances the contractile
responses of aortic segments in vitro precontracted with phenylephrine
or serotonin (5-hydroxytryptamine; 5-HT). The enhanced contractile responses elicited by each of the three inhibitor classes require an
intact endothelium and are independent of the NO pathway, i.e., they
are not influenced by inhibiting NO formation.
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MATERIALS AND METHODS |
Animals.
Sprague-Dawley strain male rats weighing ~250 g were obtained from
Charles River and maintained on a nutritionally complete pellet diet
(Purina rat chow 50001) and water ad libitum. Rats weighed 250-450
g when used in the experiments.
Preparation and incubation of aortic slices.
Rats were anesthetized rapidly by inhalation of carbon dioxide and were
then exsanguinated. The aorta was excised from its origin at the aortic
valve ring to the approximate level of the left renal artery, and the
segment, ~7.5 cm long, was chilled immediately in 145 mM NaCl-5 mM
KCl and maintained at 2°C until the onset of incubation ~10-20
min later. The adherent adventitial tissue was removed, the vessel was
cut open longitudinally and sliced into horizontal segments, and each
segment was bisected vertically for comparison of a control and
experimental sample at a given level of the aorta. Comparable slices
from 3-4 rats were usually pooled to provide sufficient tissue for
analysis. To study the formation of [14C]glutamate, the
slices were suspended in 2.5 ml of an incubation medium of the
following general composition (in mM): 118 NaCl, 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 10 D-glucose. The medium also contained unlabeled precursor,
e.g., 50 or 100 µM L-leucine, and a radioactive compound,
e.g., 0.3 µCi/ml of L-[U-14C]leucine,
specific radioactivity 314 mCi/mmol (Amersham). Suspensions were
generally shaken for 60 min at 37°C in an atmosphere of 5% CO2-95% O2. Thereafter, the tissues were
removed, blotted to remove adherent medium, weighed rapidly, and frozen
immediately in vials immersed in an ethanol-dry ice bath. Frozen
tissues were maintained at
15°C until assayed. When appropriate,
the endothelium was denuded before incubation of the slices, as
previously described (1). Tissue uptake of
[14C]leucine and estimation of the
[14C]glutamate formed were determined as described
previously (41). Uptake was quantified by the
disappearance of labeled precursor from the ambient medium and
[14C]glutamate formation by the preparation of aqueous
extracts of the tissues, resolution of the extracts by either
thin-layer chromatography and autoradiography or by anion exchange
column chromatography and quantification of the radioactive glutamate
fractions by liquid scintillation spectrometry.
[14C]Glutamate formation (expressed as nmol/g wet weight
of tissue) was calculated from radioactivity so quantified and the
specific radioactivity of the precursor or of an acetyl group of the
precursor [U-14C]leucine. As indicated by the studies
below, one two-carbon moiety of leucine is incorporated per glutamate formed.
Estimation of 14CO2 formed.
To estimate the 14CO2 evolved via the tissue
metabolism of [14C]leucine, aortic segments prepared as
described above were incubated in 2.5 ml of the following medium (in
mM): 142 NaCl, 4.7 KCl, 1.0 CaCl2, 1.2 MgSO4,
4.0 Na+ phosphate (pH 7.4), 10 D-glucose, and
appropriate unlabeled and radioactive 14C precursors.
Tissues were incubated for 1 h in closed vessels containing a KOH
trap, under an atmosphere of 100% O2, at 37°C. 14CO2 was estimated as the carbonate by liquid
scintillation counting in ScintiSafe Plus 50% counting solution
(Fisher) and the values corrected for the recovery of
14CO2, which averaged 78.8%.
These conditions were also used to assay flux across the BCDC with 100 µM
-ketoisocaproate plus
-keto-[1-14C]isocaproate
as substrate, similar to methods previously reported by others
(4, 19). We use the general term "BCDC activity" to
express the net flux assessed via 14CO2 so
evolved by segments of whole tissue. This BCDC activity is determined
by several factors, including the amount of BCDC enzyme protein, the
degree of phosphorylation of the regulatory site, i.e., the activity
state (19), the availability of the
-ketoisocaproate
substrate owing to net transamination of leucine (i.e., the forward
minus the back reaction), the transport of
-ketoisocaproate across
the mitochondrial membrane via the branched chain
-keto acid
transporter (20) and the concentration of cofactors for
the oxidative decarboxylation.
Preparation and assay of aortic homogenates.
Aortic segments were homogenized in a sucrose medium containing
antiproteases (2) and the suspensions centrifuged at 800 g for 10 min at 2°C to remove tissue clumps. The
supernatant suspensions containing ~1.0 mg/ml of protein were then
assayed for leucine-
-ketoglutarate transaminase and
alanine-
-ketoglutarate transaminase activities in 0.15 ml of the
following reaction mixture (in mM): 133 Tris buffer (pH 7.6), 0.13 pyridoxal phosphate, 6.58 Na+
-ketoglutarate, 6.58 L-leucine or L-alanine, and 0.132 µCi/ml of
-keto- [1-14C]glutarate (specific radioactivity 51.8 mCi/mmol; New England Nuclear). Reaction was initiated by addition of
homogenate containing 100 µg of protein and the mixtures incubated
with shaking at 37°C for 20 min. Thereafter, aqueous extracts were
prepared and assayed for [14C]glutamate as previously
described (41).
Estimation of glutamate.
Tissue content of free glutamate was estimated enzymatically using
glutamate dehydrogenase, as described by Lund (31).
Approximately 50-60 mg of aortic tissue were suspended and mixed
in 1.0 ml of 10% perchloric acid at 2°C for 1 h. After
centrifugation (11,500 g for 10 min) the supernatant
solutions were neutralized with KOH, insoluble potassium perchlorate
was removed by centrifugation, and the neutralized supernatants were
lyophilized. The reaction mixture for enzyme assay (total volume 1.1 ml; final pH 8.7) had the following composition: 90 mM Tris buffer, 1.8 mM EDTA, 0.57 M hydrazine, 1.5 mM
-nicotinamide adenine
dinucleotide, 0.5 mM ADP, and an appropriate aliquot of either sample,
L-glutamate standard, or water (control). Reaction at room
temperature was initiated by the addition of 200 µg (20 µl) of beef
liver glutamate dehydrogenase (120 U/mg; Boehringer-Mannheim) and the
increase in optical absorbance at 339 nm owing to the formation of
reduced nicotinamide adenine dinucleotide followed spectrophotometrically.
Contractile responses of aortic rings.
After the rats were anesthetized by inhalation of carbon dioxide and
were exsanguinated, the aorta was excised, adherent adventitial tissue
was removed, and the vessel was placed into the isotonic incubation
medium (see Preparation and incubation of aortic slices) equilibrated with 5% CO2-95% O2 at 23°C and
containing 50 µM L-leucine and 0.1 mM ascorbate. The
windkessel region, i.e., the segment from ~15-45 mm from the
aortic ring, was used, unless indicated otherwise, and 7-mm rings (mean
wet wt 10.1 ± 0.6 mg) were cut. Each ring was mounted by means of
stainless steel hooks to record isometric tension in a water-jacketed
50-ml chamber maintained at 37°C. The incubation medium described
above was equilibrated with 5% CO2-95% O2 and
recirculated through the chamber from a 300-ml reservoir at a rate of
32 ml/min. Isometric tension was recorded via a force transducer linked
to a Macintosh computer using MacLab software. The aortic segments were
mounted within 10-15 min after the death of the animal. Initial
tension was adjusted to 1.0 g and so maintained for
60-90 min. Thereafter, the recirculated medium was replaced with
fresh medium and control observation periods begun. Segments were
generally precontracted with either phenylephrine
(10
8-10
6 M) or 5-HT (5-10 µM). Test
compounds were added when plateau precontraction tension values were attained.
When indicated, the endothelium was denuded before mounting the aortic
ring by abrading with the steel supporting hooks. The procedure did not
interfere with subsequent contraction responses to phenylephrine or
5-HT, and the effectiveness of the denuding procedure was demonstrated
in each instance by the failure of 10
5 M acetylcholine to
induce a relaxation response.
Materials.
In addition to [U-14C]leucine and
-keto-[1-14C]- glutarate, the following
labeled compounds were used: 342 mCi/mmol
L-[U-14C]isoleucine, >250 mCi/mmol
L-[U-14C]- valine, 60 mCi/mmol
L-[1-14C]leucine (all from New England
Nuclear), 54 mCi/mmol 2-keto[1-14C]isocaproic acid
(Amersham), and L-[4,5-3H]leucine (50 Ci/mmol; DuPont-New England Nuclear). The following were purchased from
Sigma (St. Louis, MO): D-aspartic acid,
-ketoisovalerate (3-methyl-2-oxobutanoic acid, Na+ salt),
-ketoisocaproate (4-methyl-2-oxopentanoic acid, Na+
salt), L-leucinol (2-amino-4-methyl-1-pentanol),
L-leucinamide hydrochloride,
N
-nitro-L-arginine methyl ester
(L-NAME), l-norepinephrine bitartrate; L-phenylephrine hydrochloride, serotonin
(5-hydroxytryptamine creatinine sulfate complex), and
L-valine.
Statistical significance (P < 0.05) was evaluated by the
t-test of independent means, by the t-test of
paired comparisons, or by one-way analysis of variance. Unless
indicated otherwise, the values below are expressed as means ± SE.
 |
RESULTS |
Glutamate formation from leucine, isoleucine, and valine.
The formation of [14C]glutamate from
L-[U-14C]- leucine by sequential aortic
segments and its characteristic regional activity pattern was reported
previously (41). Identical experiments were repeated using
L-[U-14C]isoleucine or
L-[U-14C]valine and the formation of
[14C]glutamate estimated as described in
Preparation and incubation of aortic slices. Each precursor
was examined in each of 3 experiments and the results are illustrated
in Fig. 1. Glutamate formation, whether
expressed in absolute units (nmol · g wet
wt
1 · h
1) (Fig. 1A) or
as the ratio of tissue
[14C]glutamate/[14C]precursor (Fig.
1B), a parameter independent of tissue weight varies with
the extent to which each precursor is metabolized to acetoacetate and
acetyl CoA, i.e., is "ketogenic." From the known metabolic pathways
(34, 47) for catabolism of these three amino acids 5/6
carbons of leucine, 2/6 carbons of isoleucine, and 0/5 carbons of
valine are utilized for acetyl CoA formation. On the premise that
glutamate formation occurs via the acetyl CoA so formed, the relative
incorporation of 14C from leucine/isoleucine/valine is
therefore predicted to be 1.0:0.4:0.0. The areas under the curves in
Fig. 1B yield corresponding observed values of 1.0:0.41:0.02
in close agreement.

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Fig. 1.
Formation of [14C]glutamate from
[U-14C]leucine, [U-14C]isoleucine, and
[U-14C]valine by sequential segments of rat aorta in
vitro. Values are means ± SE for each of 3 experiments (9 rats)
plotted as the final tissue content of [14C]glutamate
(A) or the final tissue ratio of
[14C]glutamate to [14C]- amino acid
precursor (B). Aortic segments were prepared and incubated
for 1 h with 50 µM amino acid precursor as described in
MATERIALS AND METHODS. The values for
[14C]glutamate formed in nmol/g wet weight of tissue
(A) were calculated using the specific radioactivity of each
amino acid precursor. The values for leucine were taken from Fig. 2 of
our previous publication (41).
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Formation of 14CO2 and
[14C]glutamate.
The initial steps for metabolism of leucine are uptake into the
endothelial cells and transamination to yield
-ketoisocaproate. The
keto-acid is then oxidatively decarboxylated via the BCDC to yield
CO2 from carbon-1 and isovaleryl CoA (34, 47).
If the Leu
Glu pathway follows these steps, a
[1-14C]leucine precursor incubated with aortic segments
should yield 14CO2 but no
[14C]glutamate. Accordingly, sequential aortic segments,
both intact and denuded of endothelium, were incubated with
[1-14C]leucine in each of six experiments. No
[14C]glutamate product was detected in aqueous extracts
examined by thin-layer chromatography and autoradiography. The
evolution of 14CO2 formed from carbon-1 was
quantified, and the results are shown in Fig.
2A. Oxidative decarboxylation
of leucine follows the regional differentiation pattern characteristic
of the windkessel area and is largely eliminated by prior removal of
the endothelium. Oxidative decarboxylation as a percentage of leucine
uptake from the ambient medium by each of five sequential aortic
segments was (means ± SE) 22.4 ± 3.5, 32.8 ± 6.0, 27.8 ± 4.6, 19.1 ± 5.1, and 16.8 ± 2.8%.

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Fig. 2.
Formation of 14CO2 from
[1-14C]leucine (A) and
-keto-[1-14C]- isocaproate (B) by
sequential, intact and endothelium-denuded segments of rat aorta in
vitro. Values are means ± SE for 6 experiments (18 rats) with
leucine and 4 experiments (12 rats) with -ketoisocaproate.
Sequential aortic segments were prepared, split longitudinally, denuded
of endothelium, and were incubated for 1 h with 100 µM precursor
as described in MATERIALS AND METHODS.
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The foregoing values indicate that the regional differentiation pattern
of CO2 evolution is not due solely to differences in
leucine uptake by the endothelium. To examine the role of BCDC activity
more directly, sequential aortic segments were also incubated with the
immediate substrate for the enzyme,
-keto-[1-14C]isocaproate, in each of four experiments
and values of the 14CO2 produced are shown in
Fig. 2B. These values again follow the differentiation
pattern characteristic of the windkessel region and are largely
eliminated by prior removal of the endothelium. Expressed as a
percentage of the
-ketoisocaproate uptake, the 14CO2 evolved by five successive segments was
(means ± SE): 21.1 ± 3.4, 31.2 ± 3.4, 34.5 ± 8.2, 24.1 ± 4.9, and 16.2 ± 4.8%.
When successive aortic segments are incubated with
[U-14C]leucine, the formation of
[14C]glutamate and the evolution of
14CO2 can both be followed and the results of
three experiments are shown in Fig. 3.
These values parallel each other in the characteristic differentiation pattern of the windkessel region and each is markedly decreased by prior removal of the endothelium. Analysis of the data of
Fig. 2A and Fig. 3 leads to the following stoichiometry of
leucine utilization by the aortic endothelium of the segments studied:
each mole of leucine entering the Leu
Glu pathway via oxidative
decarboxylation by the BCDC yields as final products 1 mol of
glutamate, containing a 2-carbon moiety of leucine, plus 4 mol of
CO2. Table 1 compares the
values of three predictions based on this stoichiometry with the
corresponding observed values. The molar ratio of
[14C]glutamate-to-14CO2 formed
from [U-14C]leucine (Fig. 3) is predicted to be 0.25;
from the areas under the curves in Fig. 3 the observed value is
0.26 ± 0.02. Predicted molar ratios of
[14C]glutamate (data shown in Fig. 3) to
14CO2 formed from [1-14C]leucine
(Fig. 2A) and of 14CO2 formed from
[1-14C]leucine to 14CO2 formed
from [U-14C]leucine are also in agreement with the
observed values within experimental
error.1

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Fig. 3.
Formation of [14C]glutamate and
14CO2 from [U-14C]leucine by
sequential, intact, and endothelium-denuded segments of rat aorta in
vitro. Values are means ± SE for 3 experiments (9 rats). Aortic
segments were prepared and tested as described in the legend to Fig. 2.
Values (nmol/g wet weight of tissue) for [14C]glutamate
and 14CO2 formed, respectively, were calculated
using the specific radioactivity of a 2-carbon and 1-carbon constituent
of the precursor [U-14C]leucine. , Intact
segments; , denuded segments.
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Table 1.
Predicted and observed molar ratios of the [14C]glutamate
and 14CO2 products of aortic endothelial
metabolism of [14C]leucine in vitro
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Studies with
-ketoglutarate.
Inasmuch as the initial steps of the Leu
Glu pathway are endothelial
uptake of leucine and transamination by reaction with a suitable
-ketoacid, such as
-ketoglutarate, we studied whether the
activity of the transamination reaction is rate limiting in the
pathway. Successive aortic segments were incubated with
[1-14C]leucine in the presence and absence of 2-5 mM
-ketoglutarate and the 14CO2 produced was
quantified. Evolution of 14CO2 in four
experiments followed the activity pattern illustrated in Fig.
2A both in the absence and presence of added
-ketoglutarate. Addition of
-ketoglutarate did not increase
significantly the 14CO2 produced in the
windkessel segment, 15-60 mm from the valve ring. Transaminase
activities of homogenates prepared from five successive aortic segments
were also tested as described in Preparation and assay of aortic
homogenates by following the formation of [14C]glutamate from
-keto-[1-14C]glutarate and leucine or alanine. The
mean values observed (52.0 ± 9.3 and 75.0 ± 11.5 nmol · mg
1 · min
1,
respectively) for leucine and alanine transaminases did not vary
significantly with segment or exhibit the differentiation pattern of
the windkessel region.
The last biochemical reaction of the Leu
Glu pathway would be
conversion of
-ketoglutarate to glutamate via a transamination or
glutamate dehydrogenase reaction. We explored the possibility that the
activity of this reaction might exhibit the differentiation pattern of
the windkessel region. In each of three experiments, successive aortic
segments were incubated in the presence of 0.1 mM
-keto-[1-14C]glutarate, specific radioactivity 0.8 µCi/µmol, and the [14C]glutamate formed was
estimated. The mean (±SE) values (nmol/g wet weight) for
five successive segments, 33.7 ± 2.1, 39.5 ± 2.4, 38.9 ± 2.5, 38.3 ± 2.1, and 41.6 ± 1.7, did not differ
significantly from each other and did not exhibit the differentiation pattern.
Requirement for glucose.
Synthesis of glutamate via the Leu
Glu pathway requires a supply of
oxaloacetate for the condensation reaction with acetyl CoA, and in our
experiments D-glucose was routinely used as a source
of oxaloacetate. A requirement for the monosaccharide was demonstrated
in two experiments, in which successive aortic segments were incubated
with [U-14C]leucine in the presence and absence of 10 mM
glucose. Formation of [14C]glutamate was decreased by
67.8 ± 5.4% (means ± SE of 10 trials; P < 0.001) in the absence of glucose.
Requirement for Na+ gradient.
Endothelial cells take up leucine by both Na+-dependent and
Na+-independent transport mechanisms (32). The
following experiments demonstrate that leucine uptake dependent on a
favorable Na+ gradient extracellular/intracellular is
required for formation of glutamate via the Leu
Glu pathway.
Successive aortic segments were incubated with
[U-14C]leucine either in the control
Na+-containing medium (see Preparation and incubation
of aortic slices), in the same medium containing K+ or
Li+ in place of Na+, or in the control medium
containing in addition 1.0 mM ouabain. The formation of
[14C]glutamate was estimated and the results are
illustrated in Fig. 4. Replacement of
Na+ with K+ or Li+, respectively,
decreased [14C]glutamate formation by 93.6 ± 0.6%
(mean ± SE; 10 trials; P < 0.001) and by
61.7 ± 6.0% (5 trials; P < 0.05). Elimination of the favorable Na+ gradient by ouabain treatment
inhibited the glutamate formation by 84.0 ± 2.3% (11 trials;
P < 0.001).

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Fig. 4.
Effects of Na+ replacement or ouabain
treatment on the formation of [14C]glutamate from
[U-14C]leucine by sequential rat aortic segments in
vitro. Values are means ± SE for 5 control experiments with 15 rats (trace A), in which the segments were prepared and
incubated with 50 µM precursor leucine as described in
MATERIALS AND METHODS. Ambient medium Na+ was
replaced isosmotically with K+ in 3 experiments with 9 rats
(trace D) and with L+ in one experiment with 3 rats (trace B). Segments were tested with 1.0 mM ouabain in
3 experiments with 9 rats (trace C). Values for
[14C]glutamate formed were calculated as described in the
legend to Fig. 3.
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Replacement with K+ decreased the uptake of leucine from
the ambient medium by 34.6 ± 7.6% and reduced the
14CO2 produced by 38.6 ± 4.2%. These
values are similar to the foregoing values for oxidative
decarboxylation as a percentage of leucine uptake (see Formation
of 14CO2 and [14C]glutamate)
and indicate that ~25-40% of the leucine taken up in vitro
enters the endothelial Leu
Glu pathway.
Glutamate content of aortic segments.
The content of total free glutamate in five successive aortic segments
(originating at the aortic valve ring and each 15 mm long) was
estimated after incubation in vitro for 1 h at 37°C. The values
(µmol/g wet weight) in seven experiments (30 rats) were 0.67 ± 0.11, 1.25 ± 0.09, 1.43 ± 0.15, 0.82 ± 0.08, and
0.67 ± 0.21, respectively. Glutamate content thus exhibited a
regional pattern similar to that previously reported (41)
for the endothelial uptake of [14C]leucine and its
conversion to [14C]glutamate.
In six additional experiments (18 rats) the effects on glutamate
content of denuding the endothelium before incubation were examined. A
20-mm aortic segment from the peak area ~20-40 mm from the
aortic valve ring was split longitudinally and one-half denuded. The
glutamate contents after 1 h of incubation of the intact and
denuded segments, respectively, were 0.90 ± 0.15 and 0.25 ± 0.04 µmol/g wet weight (P < 0.005). The reduction of
~72% indicates that endothelial formation of glutamate in vitro is a
major determinant of the aortic content. In five further experiments (15 rats) aortic segments were tested similarly, except that one-half of each was denuded after incubation. The resulting glutamate values in
the intact and denuded samples, respectively, were 0.81 ± 0.14 and 0.79 ± 0.20 µmol/g, not significantly different. The results indicate that following formation in the endothelium glutamate is transferred to the underlying aortic coats. This conclusion is also
supported by studies of [14C]glutamate formation from
[14C]leucine in aortic segments denuded either before or
after incubation. Here too the [14C]glutamate content was
markedly decreased by denuding before incubation (41) but
not significantly changed by denuding after incubation.
Effects of L-leucinol.
L-Leucinol effectively inhibits the Leu
Glu pathway and
lowers the cGMP content of aortic segments in vitro (41).
The effects of 5 mM leucinol on the glutamate content and BCDC activity
of aortic segments were explored. In five experiments (15 rats), values
of the glutamate contents of control versus leucinol-treated segments,
tested as described in the preceding paragraph, were 1.17 ± 0.24 versus 0.57 ± 0.17 µmol/g (P < 0.01). The
effects of 5 mM leucinol on BCDC activity were examined by
incubating aortic segments with either
[1-14C]leucine or
-keto-[1-14C]isocaproate and estimating the
14CO2 evolved. Leucinol decreased the
14CO2 evolved from both
[1-14C]leucine (5 experiments; control and leucinol
values, respectively, 155 ± 31 and 8.6 ± 1.0 nmol/g,
P < 0.01) and
-keto-[1-14C]isocaproate (5 experiments; control and
leucinol values, respectively, 163 ± 24 and 12.1 ± 2.1 nmol/g, P < 0.005). Inhibitory effects of leucinol
could result from blocking the uptake or transamination of leucine,
e.g., by blocking the mitochondrial branched chain aminotransferase,
which functions as a branched-chain
-ketoacid transporter
(20). Direct binding to the BCDC protein is unlikely because leucinol lacks a keto group.
Other tissues.
Prior results show that the Leu
Glu pathway is present mainly in four
tissues: the aorta, pancreas, testis, and lung (41). Inasmuch as the present evidence (Figs. 2 and 3) indicates that the
differentiation of aortic segments for Leu
Glu activity is correlated
with leucine uptake and BCDC activity, it was of interest to examine
these activities in tissues differentiated or not for the Leu
Glu
pathway. Segments of 11 rat tissues were incubated with either
[U-14C]leucine, [1-14C]leucine, or
-keto-[1-14C]isocaproate and the formation of
14CO2 and [14C]glutamate
quantified. The results listed in Table 2
confirm a high correlation (
= 0.97) between
[14C]glutamate formation and
14CO2 evolution from
[U-14C]leucine. The four tissues with an active Leu
Glu
pathway all exhibit relatively high leucine uptake and BCDC activity
assessed by 14CO2 evolved from
-ketoisocaproate. The remaining tissues have little or no Leu
Glu
activity, owing to a number of patterns. The liver, kidney, and heart
exhibit much reduced leucine uptake and metabolism to CO2,
although the BCDC activity is high. The jejunum, and less markedly the
thymus, have relatively little BCDC activity but take up leucine well.
Skeletal muscle and cerebral cortex exhibit little uptake or BCDC
activity.
Contractile responses: effects of L-leucinol and
L-leucinamide.
As indicated above, L-leucinol is an effective inhibitor of
the Leu
Glu pathway. The effects of leucinol (0.2-5.0 mM) on the contractile responses of segments precontracted with phenylephrine or
5-HT are shown in Figs.
5 and 6. Figure 5, A and
B, illustrates prominent increases in tension owing to
treatment with 5.0 mM leucinol of individual segments precontracted,
respectively, with 10
7 M phenylephrine or 5 µM 5-HT.
Figure 5, A' and B', shows the marked attenuation
of these responses in corresponding, adjacent segments denuded of
endothelium and treated similarly. The peak increments in tension owing
to leucinol were proportional to the concentration tested in the range
of 0.2-5.0 mM, as shown in Fig. 6,
and the values were similar for segments precontracted with either
10
7 M phenylephrine (10 rats, 24 trials,
F = 43.6, P < 0.005) or 5 µM 5-HT (8 rats, 12 trials, F = 23.6, P < 0.005).
The control, mean tension value before addition of 5 mM leucinol,
312 ± 22 g/g wet wt, was increased by ~264 g/g wet wt, or
84.6%, by leucinol treatment. When four additional segments (2 rats)
were denuded of endothelium and tested similarly, 5 mM leucinol
elicited a much smaller increment, 50 ± 24 g/g, significantly
less than the increment observed with intact segments
(t = 4.66, P < 0.001).

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Fig. 5.
Contractile responses of individual intact and
endothelium-denuded rat aortic segments to inhibitors of the
leucine-to-glutamate pathway. Each inhibitor was tested with an
intact (A-F) and an adjacent denuded
(A'-F') segment. Segments were precontracted at
the time point indicated by arrow 1 and test inhibitor added at arrow
2. A,A': precontracted with 10 7 M
phenylephrine and tested with 5 mM L-leucinol;
B,B': precontracted with 5 µM
5-hydroxytryptamine (5-HT) and tested with 5 mM L-leucinol;
C,C': precontracted with 10 µM 5-HT and tested
with 10 mM L-leucinamide; D,D':
precontracted with 10 7 M phenylephrine and tested with 10 mM -ketoisovalerate; E,E': precontracted with
10 6 M phenylephrine and tested with 2 mM
L-valine; F,F': precontracted with
10 6 M phenylephrine and tested with 20 mM
D-aspartate.
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Fig. 6.
Increments in tension owing to treatment of precontracted
rat aortic segments with various concentrations of
L-leucinol. Values are means ± SE for segments
precontracted with 10 7 M phenylephrine (trace
A) or 5 µM 5-HT (trace B). Mean tension of the
precontracted segments before additon of leucinol was 312 ± 22 g/g.
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Two experimental approaches were adopted to examine whether leucinol
increases contractility by inhibiting endothelial NO formation. Aortic
segments precontracted with 10
7 M phenylephrine were
tested for relaxation responses to 10
5 M acetylcholine in
the absence and presence of 1 or 5 mM leucinol. Values of the percent
relaxation observed in six control versus four treated segments,
64.8 ± 6.2% versus 66.6 ± 6.3%, respectively, were not
significantly different. The effects of 5 mM leucinol on segments
precontracted as above were also examined in the absence and presence
of 2 mM L-NAME added to inhibit NO formation. Whereas L-NAME abolished the relaxation responses to acetylcholine,
it did not alter significantly the leucinol-induced increments in tension, with observed values for control versus
L-NAME-treated segments of 230 ± 66 versus 170 ± 53 g/g (4 trials, P > 0.40).
L-Leucinamide (2-10 mM) also increased the contractile
responses of segments precontracted with 10 µM 5-HT. In seven trials (5 rats) treatment with 10 mM leucinamide yielded tension increments of
291 ± 34 g/g (t = 8.64, P < 0.001), a mean increase of 130%. When four segments were denuded of
endothelium and tested similarly, the leucinamide-induced increment was
decreased by ~79% to 61 ± 32 g/g (t = 4.50, P < 0.005). The effects of leucinamide on individual
segments are illustrated in Fig. 5C (endothelium intact) and
Fig. 5C' (endothelium denuded).
Contractile responses: effects of
-ketoisovalerate and
L-valine.
The catabolism of L-valine is initiated by its conversion
to
-ketoisovalerate, which is then oxidatively decarboxylated by the
same BCDC, which acts on
-ketoisocaproate derived from leucine (36). Inasmuch as the valine and
-ketoisovalerate
metabolic pathway does not yield acetyl CoA or glutamate, these
compounds will competitively inhibit the functions of the Leu
Glu
pathway by preempting the BCDC. Accordingly, their effects on aortic
contractile responses were examined in segments precontracted with
phenylephrine, and the results are listed in Table
3 and illustrated for individual segments
in Fig. 5, D,D' and E,E'. In tests of 17 segments
(10 rats) precontracted with 10
7 M phenylephrine,
treatment with 10 mM
-ketoisovalerate increased the tension values
by 101 ± 12 g/g (P < 0.001), or ~42% on
average, and similar increments were observed in 9 additional trials (4 rats) of segments precontracted with 10
6 M phenylephrine
(P < 0.001). By contrast, five segments (3 rats) denuded of endothelium showed no significant change in tension on
treatment with 10 mM
-ketoisovalerate (P < 0.001 for difference from intact segments), although they contracted
normally on exposure to 10
7 M phenylephrine (Table 3). In
tests of 16 segments (6 rats) precontracted with 10
6 M
phenylephrine, treatment with 2 mM L-valine increased the
tension values by 45 ± 6 g/g (P < 0.001), or
~16% on average, and significant increments (P < 0.001) were also observed in eight further trials (4 rats) after
precontraction with 10
7 M phenylephrine. Again, denuding
the endothelium of three segments (2 rats) eliminated the effect of
valine (P < 0.001 for difference from intact
segments), although the contractile responses to phenylephrine were
intact (Table 3).
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Table 3.
Effects of -ketoisovalerate and L-valine on the tension
of intact or endothelium-denuded aortic segments precontracted with
phenylephrine
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Inasmuch as BCDC activity exhibits the characteristic aortic pattern of
peak activity just distal to the aortic arch and decreasing activity
distally, we tested whether increases in contractile responses owing to
preemption of the enzyme by
-ketoisovalerate or valine follow a
similar pattern. Successive aortic segments were tested as described
above with 10 mM
-ketoisovalerate or 2 mM valine, and the increments
in tension were plotted versus mean distance from the aortic valve
ring. As shown in Fig. 7, the patterns
are similar to that observed for BCDC activity: highest values in the
segments ~12-26 mm from the aortic ring, just distal to the
arch, and decreasing values distally.

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Fig. 7.
Increments in tension of successive aortic segments
precontracted with phenylephrine and treated with 10 mM
-ketoisovalerate (13 rats, 36 segments) or 2 mM
L-valine (9 rats, 29 segments). Values are means ± SE. Segments 7-mm long were precontracted and tested as described in
Table 3. Values for tension increments were plotted at the midpoint of
each segment tested. The aortic arch, 0-12 mm from the aortic
ring, was not tested.
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The increments in tension observed with
-ketoisovalerate do not
result from inhibition of the endothelial NO pathway. Values of the
percent relaxation of six control segments precontracted with
10
6 M phenylephrine and treated with 10
5 M
acetylcholine, 64.8 ± 6.2%, were not significantly different from those of six adjacent segments tested similarly in the presence of
10 mM
-ketoisovalerate, 70.1 ± 4.4%. Furthermore, the
presence of 2 mM L-NAME to block NO formation did not
decrease responses to
-ketoisovalerate. The increments in tension
owing to
-ketoisovalerate in two control and two adjacent
L-NAME-treated segments, respectively, were 107 ± 0.28 and 163 ± 0.06 g/g.
-Ketoisovalerate also enhanced the contractile responses of segments
precontracted with norepinephrine or 5-HT. In tests of four segments
precontracted with 10
7 M norepinephrine, the addition of
10 mM
-ketoisovalerate yielded tension increments of 81.8 ± 21.1 g/g (P < 0.025). In tests of six pairs of
adjacent segments precontracted with 10 µM 5-HT, 10 mM
-ketoisovalerate increased both the duration of the contractile response (controls, 79 ± 7 min; treated, 107 ± 10 min;
P < 0.01) and the total response, i.e., the area under
the response curve (controls, 80.8 ± 21.1 g/min; treated,
127.3 ± 31.8 g/min; P < 0.02).
Segments precontracted with 10
7 M phenylephrine were
further tested with
-ketoisocaproate, the intermediate of the
Leu
Glu pathway. Although similar in chemical structure to
-ketoisovalerate, 5-10 mM
-ketoisocaproate did not
significantly increase tension values in five tests but did eliminate
contractile responses to 5-10 mM
-ketoisovalerate in four trials.
Contractile responses: effects of D-aspartate.
Glutamate formed via the endothelial Leu
Glu pathway is transported
to the underlying smooth muscle coats. Interruption of the Leu
Glu
pathway by D-aspartate, a relatively specific and metabolically inert inhibitor of Na+-dependent glutamate
transporters (17, 28, 42), also enhanced contractile
responses. Fifty-two segments (9 rats) precontracted with
10
6 M phenylephrine were tested with various
concentrations of D-aspartate, from 2-20 mM. Figure 5,
F and 5F' illustrates responses of an intact and
an endothelium-denuded segment. Figure 8
shows that increments in tension were observed throughout the
concentration range tested (F = 11.36, P < 0.005) and increased from 44 ± 6 g/g
(~15.6%) at 2.0 mM to 131 ± 11 g/g (~46.5%) at 20 mM. In
five segments denuded of endothelium 20 mM D-aspartate
treatment increased the tension by only 21 ± 2.9 g/g (Fig. 8), a
decrease of ~84% compared with the intact segments
(P < 0.001). Treatment of two segments with 2 mM
L-NAME, followed by 20 mM D-aspartate, yielded tension increments of 131 ± 2 g/g, similar to the control values. Precontraction of six segments with 10 µM 5HT, followed by 2-20 mM D-aspartate, yielded tension increments comparable to
the foregoing values.

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Fig. 8.
Effects of various concentrations of
D-aspartate on the increase in tension of precontracted rat
aortic segments. Values are means ± SE for 52 trials (36 segments, 9 rats). Segments were precontracted with 10 6 M
phenylephrine, and the mean tension before addition of
D-aspartate was 282 ± 21 g/g. Corresponding
increments in tension for 5 segments (2 rats) denuded of endothelium
and tested similarly with 20 mM D-aspartate are shown; mean
tension before D-aspartate was 337 ± 40 g/g.
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Na+ L-cysteate, another competitive inhibitor
of glutamate transport, also elicited contractile responses. On
addition of 20 mM L-cysteate to three aortic segments
precontracted with 10
6 M phenylephrine, the initial
tensions, 219 ± 13 g/g, increased to 420 ± 54 g/g
(P < 0.01).
Contractile responses: effects of external L-glutamate.
Because inhibition of the Leu
Glu pathway enhances contractile
responses, glutamate added externally is expected to induce relaxation
responses, providing it permeates the Leu
Glu compartment. The
permeation, however, is poor (41), and addition of
0.2-5.0 mM L-glutamate to aortic rings precontracted
with 10
6 M phenylephrine yielded no consistent change in
tension. Raising the glutamate concentration to 10-20 mM, however,
produced a biphasic response. In tests of 11 segments (4 rats) so
treated versus 7 untreated adjacent segments, external glutamate
elicited a rapid increase in tension (from initial values of 411 ± 28 to 506 ± 17 g/g), which persisted for 39 ± 4 min and
was followed by a prolonged relaxation. The glutamate-treated segments
relaxed at a rate of 2.30 ± 0.28 g · g
1 · min
1 compared with
a slower rate in the untreated segments, 0.71 ± 0.12 g · g
1 · min
1
(P < 0.001). The relaxation responses to glutamate
were similar and significant whether the aortic segments were
precontracted with 10
6 M phenylephrine alone (4 trials,
P < 0.01), or with phenylephrine, followed by either
20 mM D-aspartate (3 trials, P < 0.025) or 20 mM Na+ cysteate (4 trials, P < 0.0125).
Denuding the endothelium had no significant effect on the responses to glutamate.
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DISCUSSION |
The results above demonstrate that the aortic Leu
Glu pathway
proceeds via the catabolism of leucine to acetyl CoA and acetoacetate, a metabolic route established by studies of other tissues (12, 34, 47). In the rat aorta, this pathway comprises uptake of L-leucine at the luminal membrane of the endothelial cell
by a Na+-dependent mechanism (Fig. 4); transamination to
-ketoisocaproate, either in the cytosol or after transport across
the mitochondrial membrane (20); oxidative decarboxylation
to isovaleryl CoA by the BCDC of the mitochondrial matrix; successive
dehydrogenation, carboxylation and hydration reactions to yield HMG
CoA; and cleavage of HMG CoA to acetyl CoA and acetoacetate. In the
presence of oxaloacetate, derived in our experiments from glucose
metabolism, the acetyl CoA can be used to synthesize
-ketoglutarate
via tricarboxylic acid cycle reactions and this product converted to
glutamate by transamination or a glutamate dehydrogenase reaction. As a
consequence of the catabolic pathway [1-14C]leucine does
not yield labeled glutamate, owing to the loss of carbon 1 in the BCDC
reaction, and the relative efficiency of formation of
[14C]glutamate from either [U-14C]leucine,
[U-14C]- isoleucine, or [U-14C]valine
(Fig. 1), respectively, corresponds to the number of labeled carbon
atoms in each amino acid available for acetyl CoA formation, i.e.,
5:2:0.
The efficiency of conversion of leucine to glutamate, the stoichiometry
of the process and the controlling role of the BCDC reaction were
characterized by quantification of the 14CO2
evolved from the metabolism of [1-14C]leucine,
-keto-[1-14C]isocaproate, and
[U-14C]- leucine. As shown in Figs. 2 and 3, leucine
metabolism to CO2 occurs primarily in the endothelium and
follows the regional differentiation pattern characteristic of the
Leu
Glu pathway. In the segments of peak activity ~25-40% of
the leucine taken up from the ambient medium was oxidatively
decarboxylated, and for each mole so metabolized there resulted 1 mol
of glutamate plus 4 mol of CO2 (Table 1). Aortic segments,
therefore, are relatively efficient at utilizing acetyl CoA derived
from leucine for glutamate formation. One mechanism accounting for the
efficiency is the relatively direct utilization for glutamate
synthesis of the
-keto-[14C]glu- tarate formed
from [U-14C]leucine-derived acetyl CoA. Such
-keto-[14C]glutarate would be labeled at carbons 4 and
5 (34), as would the [14C]glutamate formed
directly from it by transamination or the glutamate dehydrogenase
reaction. If the
-keto-[14C]glutarate entered the
tricarboxylic acid cycle, however, the 14C would be
randomized in the succinate and fumarate intermediates and label
carbons 1-4 of
-ketoglutarate and glutamate derived from it.
The extent of such randomization can be assessed by determining the
14C content of carbon-1 in glutamate. In a prior study (41)
[14C]glutamate formed via the Leu
Glu pathway was
treated with L-glutamate decarboxylase and the carbon 1 evolved as CO2 contained insignificant 14C.2 Thus the
-ketoglutarate intermediate was converted to glutamate relatively
directly. The mechanisms responsible are unknown but may involve
compartmentalization in mitochondria or other organelles.
The Leu
Glu, NO, and PGH2 signal pathways share a similar
regional pattern of activity in the rat aorta (1, 40, 41), suggesting that the windkessel area, site of the maximal activity, is
functionally specialized for regulating contractility of the underlying
smooth muscle. Hence we sought a controlling reaction in the leucine
catabolic sequence whose activity would correspond to the Leu
Glu
regional pattern. We focused on the BCDC reaction, which controls
leucine catabolic flux in a number of other tissues. As shown in Fig.
2B, endothelium-dependent oxidative decarboxylation of
-keto-[1-14C]isocaproate by the BCDC in successive
aortic segments clearly exhibits the regional pattern. Furthermore, we
calculated the extent to which the differences in endothelium-dependent
BCDC activity between the five successive segments shown in Fig.
2B could account for the corresponding segment-dependent
differences in overall Leu
Glu activity, i.e., glutamate formation
(Fig. 3). Let a represent values of the differences between
the mean, endothelium-dependent BCDC activities of the successive
aortic segments, estimated as the 14CO2
produced from
-keto-[1-14C]isocaproate and expressed
relative to the peak 14CO2 producton (Fig.
2B). Let b represent the corresponding values for
the differences between the mean, endothelium-dependent Leu
Glu activities of the successive aortic segments, estimated as the glutamate produced and expressed relative to the peak glutamate production (Fig. 3). The ratio b/a then expresses
the dependence of the segmental differences in overall Leu
Glu
activity on the differences in BCDC activity, and a value of 1.0 represents complete dependence (22). The observed
b/a values for four segmental differences
(means ± SE) are 1.01 ± 0.06, in accord with complete dependence on the BCDC activity.
In contrast with the close correspondence of the Leu
Glu and BCDC
regional patterns of activity, no such pattern was observed for
leucine-
-ketoglutarate or alanine-
-ketoglutarate transaminase activities in aortic homogenates of successive segments. Additon of
-ketoglutarate to the incubation medium composed of segments incubated with [1-14C]leucine yielded no increase in
14CO2 production and no change in the regional
pattern shown in Fig. 2A, consistent with the conclusion
that leucine-
-ketoglutarate net transamination to yield
-ketoisocaproate is not rate limiting in these segments. Two distal
reactions of the leucine catabolic pathway also showed no regional
segmental differences. Incubation of successive aortic segments with
1.7 µM [9,10-3H]oleate as a source of acetyl CoA in
place of leucine yielded [3H]glutamate averaging
2.05 ± 0.26 nmol/g wet weight with no significant segmental
differences. Incubation with
-keto-[1-14C]glutarate as
precursor yielded [14C]glutamate averaging 39 ± 2 nmol/g and no significant segmental differences. The evidence,
therefore, favors the BCDC reaction as the determinant of the regional
activity pattern of the Leu
Glu pathway. Specific factors that
influence this reaction and could determine the regional pattern
include the amount of BCDC enzyme protein, the degree of
phosphorylation determining the activity state of the enzyme
(19), the availability of the
-ketoisocaproate substrate owing to net transamination of leucine, the transport of
-ketoisocaproate across the mitochondrial membrane via the branched
chain
-ketoacid transporter (20), and the concentration of cofactors required for the oxidative decarboxylation.
Estimations of the total free glutamate content of aortic segments
incubated in vitro indicate that the endothelial Leu
Glu pathway is a
major determinant of that content. Thus the segmental values exhibit
the characteristic regional pattern of the pathway, and in the peak
segment (~20-40 mm from the aortic valve ring) denuding the
endothelium before incubation decreased the content by ~72%.
Moreover, incubation with the Leu
Glu pathway inhibitor L-leucinol decreased the value by ~51%. When the
segments were denuded of endothelium after incubation, the underlying
coats retained the glutamate. Similarly, denuding the endothelium
before incubation markedly reduced the formation of
[14C]glutamate from [U-14C]leucine
(41), whereas denuding after incubation did not alter the
total [14C]glutamate content of the segment. These
observations indicate that the final steps of the Leu
Glu pathway
involve efficient transport to the underlying coats. The transfer
mechanism(s) responsible has not been characterized, but the transport
route is highly compartmentalized and bounded by structures relatively
impermeant to glutamate, inasmuch as no [14C]glutamate
was detected in the ambient media after incubation of segments with
[U-14C]leucine (41).
Leucine catabolism has been studied extensively in nonaortic tissues in
relation to energy metabolism (12, 14), protein synthesis
and degradation (5, 37), and familial disorders such as
maple syrup urine disease (11, 27) and isovaleric acidemia
(48), which result from genetic defects in the BCDC and
isovaleric dehydrogenase, respectively. Normal diets provide the human
and the rat with an excess of the branched-chain amino acids,
considerably more than is required for new protein synthesis in both
growing and nongrowing individuals (14). The leucine catabolic pathway therefore functions in nonaortic tissues as an
essential disposal route for potentially toxic branched-chain intermediates, simultaneously providing calories for immediate use or
storage as adipose tissue (14). Several variables,
including starvation (13, 19), dietary protein level
(19), diabetes mellitus (3), octanoate
infusion (4), and insulin and growth hormone
(14) modulate flux through the pathway, with the direction of change dependent on the particular tissue. Generally, the
controlling enzyme in these responses is the BCDC, whose activity is
decreased by covalent phosphorylation and increased by
dephosphorylation (12, 19). In rat aortic endothelium the
leucine catabolic pathway is utilized to supply acetyl CoA for
glutamate synthesis and transfer to the underlying smooth muscle.
We tested the hypothesis that the Leu
Glu pathway, along with the NO
and PGH2 pathways, functions to regulate the contractility of the aortic smooth muscle. Because leucinol and leucinamide interrupt
the pathway and decrease the cGMP content of the smooth muscle, we
predicted they would enhance aortic contractile responses, and the
results above confirm the prediction. The enhanced responses require an
intact endothelium and do not occur via reductions in NO formation or
action. The specificity of action of these compounds is an important
consideration because millimolar concentrations are required.
Consequently we also tested two additional classes of Leu
Glu pathway
inhibitors, compounds that are chemically different from the leucine
analogs and from each other.
-Ketoisovalerate and its precursor
L-valine were chosen to preempt competitively the
endothelial BCDC. These compounds, too, act on the endothelium to
enhance contractile responses and the effects exhibit a regional aortic
pattern (Fig. 7) similar to that of the BCDC activity (Fig. 2).
Specificity for the interruption of the Leu
Glu pathway at the BCDC
site is also indicated by the observations that
-ketoisocaproate, the BCDC substrate derived from leucine, does not enhance contractile responses but does block the inhibition owing to
-ketoisovalerate.
-Ketoisovalerate produced contractile responses in the presence of
L-NAME and did not interfere with acetylcholine-induced
relaxations. Hence the effects of
-ketoisovalerate are not mediated
by blocking NO formation or action.
Interrupting the Leu
Glu pathway by treatment with
D-aspartate, a metabolically inert compound that competes
with L-glutamate for transfer mediated by certain glutamate
transporters, also causes enhanced contractile responses. These effects
require an intact endothelium and do not result from reductions in NO
formation. The results point to the involvement of glutamate
transporters in the terminal steps of the pathway in which glutamate is
transferred efficiently to the underlying coats without significant
exchange with the extracellular medium. The mechanisms, membrane
transporters, and routes responsible for this compartmentalized
transport are unknown at present, and the precise locus of action of
D-aspartate in the endothelium remains to be determined. In
studies of nonaortic tissues, transport mechanisms with specificity for
both L-glutamate and D-aspartate have been
found in mitochondrial (29) and plasma membranes
(10). The X
family of glutamate/D-aspartate transporters (10), which
mediate Na+-dependent transport across plasma membranes,
have been particularly well characterized and a number cloned
(43, 46). These function prominently, for example, in
glial and neuronal cells of the central nervous system where efficient
cellular uptake of glutamate is essential to maintain low extracellular
concentrations and prevent indiscriminate signaling and nerve damage
(17). It is reasonable to suggest that the
compartmentalized transport of glutamate synthesized in the aortic
Leu
Glu pathway is also necessary for discrete signaling, particularly because free glutamate is relatively abundant in blood and
many tissues.
Relatively high concentrations, 10-20 mM, of externally added
glutamate elicited a rapid-onset increase of ~23% in tension followed, after a delay of ~39 min, by a prolonged relaxation response. The high concentrations required and the slow onset of the
relaxation responses could result from the relative impermeability to
glutamate of the Leu
Glu pathway. In prior studies glutamate treatment (0.5-2.0 mM) of rat aortic segments did increase cGMP levels but only when the segments were denuded of endothelium and
treated with 3-isobutyl-1-methylxanthine to inhibit cyclic nucleotide
phosphodiesterase (41). Precontracted rabbit aortic rings
were reported to exhibit relaxation responses on treatment with 100 mM
glutamate or 1-10 mM glutamine (33).
Systematic studies (1, 40, 41) of sequential rat aortic
segments in vitro provide comprehensive evidence of regional differentiation of the aortic endothelium. Figure
9 illustrates the patterns observed for
six activities whose values peak in the windkessel area and decrease
distally: constitutive formation of NO from L-arginine
(1), cGMP content (1), and increase in cGMP
owing to inhibition of the cyclooxygenase arm of the eicosanoid pathway
and PGH2 formation (40), endothelial uptake of
[U-14C]leucine and its conversion to
[14C]glutamate (41), free glutamate content,
and BCDC activity (Fig. 2). The increase in values from the aortic arch
(first ~12 mm from the valve ring) to the windkessel area are
noteworthy in view of the ultrastructural studies utilizing
morphometric methods reported by Kao et al. (23). They
observed that rat endothelial cells in the aortic arch are longer and
thinner and contain fewer intracytoplasmic vesicles and fewer complex
interdigitating intercellular contacts than those in the windkessel
area.

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Fig. 9.
Patterns of regional differentiation in rat aorta for six
activities dependent on the endothelium. For comparison, data are
plotted as percentages of the maximal value observed for each activity.
Trace A, formation of [14C]glutamate from
[14C]leucine (41); trace B,
tissue content of free glutamate; trace C, tissue
cGMP levels (1); trace D, BCDC enzyme activity
assayed by the liberation of 14CO |
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