J Appl Physiol
91: 231–238, 2001.
Purine salvage to adenine nucleotides in different
skeletal muscle fiber types
JEFFREY J. BRAULT AND RONALD L. TERJUNG
Department of Physiology, College of Medicine, Department of Biomedical
Sciences, College of Veterinary Medicine, and Dalton Cardiovascular
Research Center, University of Missouri, Columbia, Missouri 65211
Received 12 December 2000; accepted in final form 16 February 2001
THE TOTAL ADENINE NUCLEOTIDE (AdN ⫽ ATP ⫹ ADP ⫹
AMP) content in skeletal muscle is controlled by three
processes: purine degradation, which establishes a loss
of AdN, and purine de novo synthesis and purine salvage pathways, which produce AdN. In order for the
AdN pool to be maintained, the rate of degradation
must balance the rate of production. For example,
degradation of AdN to purine nucleosides and bases
increases during exercise and is most extreme during
intense contractions (2, 15, 17, 19, 32, 37), a process
exacerbated by ischemia (2, 37). The rates of de novo
synthesis and/or purine salvage must be increased to
maintain the AdN pool after these challenges.
Nucleotide degradation to nucleosides occurs via the
enzyme 5⬘-nucleotidase by dephosphorylation of the
ribose of AMP and IMP to form adenosine and inosine,
respectively (36). The purine nucleotide IMP is produced from AMP by the enzyme AMP deaminase. Compared with 5⬘-nucleotidase, the activity of AMP deaminase is dominant in skeletal muscle and can result in
large accumulations of IMP (2, 25, 44). Thus nucleotide
degradation is initiated primarily by the production of
IMP. Inosine may be further degraded to its purine
base hypoxanthine by the action of purine nucleoside
phosphorylase. Although nucleotides do not readily
leave the cell, purine nucleosides (adenosine and inosine) and bases (primarily hypoxanthine) are able to
cross the cell membrane and escape the myocyte. Numerous reports indicate that inosine and, particularly,
hypoxanthine efflux occurs to a marked degree in humans after intense exercise (15, 33). In fact, repeated
days of intense exercise lead to an apparent deficit in
AdN in the rested muscle (16, 19, 32). This implies that
the processes of AdN production may not occur at a
rate sufficient to return the AdN to normal before the
next day’s exercise bout.
It is recognized that the rates of de novo synthesis of
AdN are low in skeletal muscle (30, 38), although there
are significant differences among muscle fiber phenotypes (34, 35). De novo synthesis rates range between
25 and 60 nmol 䡠 h⫺1 䡠 g muscle⫺1, representing fractional synthesis rates of 0.3–1.0% of the AdN pool per
hour (34). In contrast, the absolute rates of AdN synthesis in skeletal muscle via the salvage pathway in
vivo are not known. Studies in cultured muscle cells
(7, 43) and intact skeletal muscle (24, 42) demonstrate that adenine and hypoxanthine can be incorporated into the AdN pool via the actions of adenine
Address for reprint requests and other correspondence: R. L.
Terjung, Biomedical Sciences, College of Veterinary Medicine, E102
Vet Med Bldg., University of Missouri, Columbia, MO 65211 (E-mail:
[email protected]).
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.
adenine; hypoxanthine; ribose
http://www.jap.org
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society
231
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Brault, Jeffrey J., and Ronald L. Terjung. Purine
salvage to adenine nucleotides in different skeletal muscle
fiber types. J Appl Physiol 91: 231–238, 2001.—Rates of
purine salvage of adenine and hypoxanthine into the adenine
nucleotide (AdN) pool of the different skeletal muscle phenotype sections of the rat were measured using an isolated
perfused hindlimb preparation. Tissue adenine and hypoxanthine concentrations and specific activities were controlled
over a broad range of purine concentrations, ranging from 3
to 100 times normal, by employing an isolated rat hindlimb
preparation perfused at a high flow rate. Incorporation of
[3H]adenine or [3H]hypoxanthine into the AdN pool was not
meaningfully influenced by tissue purine concentration over
the range evaluated (⬃0.10–1.6 ␮mol/g). Purine salvage
rates were greater (P ⬍ 0.05) for adenine than for hypoxanthine (35–55 and 20–30 nmol 䡠 h⫺1 䡠 g⫺1, respectively) and
moderately different (P ⬍ 0.05) among fiber types. The lowoxidative fast-twitch white muscle section exhibited relatively low rates of purine salvage that were ⬃65% of rates in
the high-oxidative fast-twitch red section of the gastrocnemius. The soleus muscle, characterized by slow-twitch red
fibers, exhibited a high rate of adenine salvage but a low rate
of hypoxanthine salvage. Addition of ribose to the perfusion
medium increased salvage of adenine (up to 3- to 6-fold, P ⬍
0.001) and hypoxanthine (up to 6- to 8-fold, P ⬍ 0.001),
depending on fiber type, over a range of concentrations up to
10 mM. This is consistent with tissue 5-phosphoribosyl-1pyrophosphate being rate limiting for purine salvage. Purine
salvage is favored over de novo synthesis, inasmuch as delivery of adenine to the muscle decreased (P ⬍ 0.005) de novo
synthesis of AdN. Providing ribose did not alter this preference of purine salvage pathway over de novo synthesis of
AdN. In the absence of ribose supplementation, purine salvage rates are relatively low, especially compared with the
AdN pool size in skeletal muscle.
232
PURINE SALVAGE IN SKELETAL MUSCLE
MATERIALS AND METHODS
Animals
Male Sprague-Dawley rats (Taconic Farms, Germantown,
NY and Harlan, Indianapolis, IN) weighing 375–400 g were
housed two or three animals per cage with unrestricted food
(Purina rat chow) and water. Living quarters were maintained at 20°C with a 12:12-h light-dark cycle. This study
was approved by the University of Missouri-Columbia Animal Care and Use Committee.
Hindquarter Perfusion
Perfusion medium. The perfusion medium, prepared on
the morning of the experiment, consisted of 5% bovine albumin in Krebs-Henseleit buffer, 5 mM glucose, 100 ␮U/ml
insulin, and typical plasma concentrations of amino acids (4).
Immediately before use, this solution was filtered and
warmed to 37°C. A portion was used to prime the perfusion
apparatus, which included, in series, a peristaltic pump, a
filter, a gas-exchange chamber supplied with 95% O2-5% CO2
for oxygenation and pH control to 7.4, and a bubble trap. The
entire apparatus was located inside a Plexiglas cabinet maintained at 37°C. Perfusion pressure and temperature were
monitored continuously throughout the experiment.
Surgical preparation. Rats were anesthetized with pentobarbital sodium (60 mg/kg ip), and 100% O2 was adminis-
tered during preparation for hindquarter perfusion as developed previously (13). After the right carotid artery was
catheterized, the testes, bladder, seminal vesicles, prostate,
large intestine, and small intestine were removed, exposing
the descending aorta and inferior vena cava. The hindfeet
and tail were tied tightly with umbilical tape, and the animal
was then placed in the warmed cabinet. With the pump
circulating perfusate at an initial low flow rate (⬃3 ml/min),
an inflow catheter (16 gauge) was secured in the descending
aorta and an outflow catheter (14 gauge) was secured in the
vena cava. The rat was then killed with an overdose of
pentobarbital sodium into the carotid artery. To clear most of
the rat red blood cells, the initial 150 ml of venous effluent
were discarded; then the perfusate was recirculated. This
yielded a hematocrit of ⬍1%, thereby avoiding significant
purine salvage by the red blood cells (26).
Perfusion protocol. The perfusion flow rate was increased
over 20–25 min to 50 ml/min. Aortic artery perfusion pressure was ⬃45 mmHg. This establishes a flow rate of ⬃0.6
ml 䡠 min⫺1 䡠 g perfused tissue mass⫺1 (13). During this time, a
fresh 300-ml volume of perfusate was prepared with either
adenine (0.3–3.0 mM) or hypoxanthine (0.4–4.2 mM) and the
respective [2,8-3H]purine base (Moravek Biochemicals) at a
specific activity of 600–2,900 dpm/nmol. When appropriate to
the design of the experiment, D-ribose was included in the
perfusion medium. The experiments that measured purine
de novo synthesis rates also included 1 ␮Ci/ml perfusate
[U-14C]glycine (Moravek Biochemicals) at a glycine concentration of 0.38 mM, which is typical for rat plasma (4). The
perfusate was switched to that containing labeled substrate
after the perfusion pressure was stabilized. This defined time
0 for the experiment.
After 30 min of perfusion with the labeled purine base, the
right lower hindlimb muscle sections were quick-frozen using
aluminum tongs cooled in liquid nitrogen. Sections included
the soleus (predominantly slow-twitch red fibers), plantaris
(mixed fast-twitch fibers), superficial medial white gastrocnemius (predominantly fast-twitch white fibers), deep lateral
red gastrocnemius (predominantly fast-twitch red fibers),
and the remainder of the gastrocnemius (mixed fast-twitch
fibers) (3). The right iliac artery was immediately ligated,
and the musculature above the knee was tightly tied with
umbilical tape to close the vascular system. The flow was
then reduced to ⬃40 ml/min to yield the identical perfusion
pressure and establish the same flow per gram of muscle as
that before muscle collection. At 60 min, the perfusate was
switched to identical medium without either purine base or
radioactivity to evacuate the precursor from the vascular
space. Timed samples of the venous effluent were initially
collected to verify the effective removal of perfusate radioactivity. After 5 min without recirculation, the left lower hindlimb muscle sections were collected in a manner identical to
that used to collect sections from the right side. Frozen tissue
samples were stored at ⫺80°C until analyzed.
Analyses
Metabolites from muscle sections were extracted in cold
3.5% (wt/vol) ethanolic (20% vol/vol) perchloric acid and neutralized with tri-n-octylamine and 1,1,2-trichlorotrifluoroethane (8). Perfusate samples were similarly extracted using
perchloric acid. Extracts were stored at ⫺80°C until analyzed.
Purine nucleotides, nucleosides, and bases were separated
using reverse-phase high-performance liquid chromatography (HPLC) and quantified by comparing peak area at 254
nm with known standards (37). ATP, ADP, IMP, hypoxan-
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5-phosphoribosyl-1-pyrophosphate (PRPP) transferase
(APRT) and hypoxanthine/guanine PRPP transferase
(HPRT), respectively. Absolute rates of purine salvage
were measured using isolated muscle in vitro, where
the incubation medium could be controlled (38). Purine
salvage appears to be favored over de novo synthesis
(30, 41, 43) and is increased significantly in muscle
with ribose supplementation (7, 14, 24, 42) likely because of increased availability of PRPP. Thus purine
salvage can be expected to contribute significantly to
AdN synthesis. However, the rates of purine salvage
have not been determined, especially in the fast-twitch
low-oxidative fiber type, which is most likely to produce
IMP during intense muscle contractions (22).
The purpose of the present study was to directly
measure purine salvage rates among skeletal muscle
fiber types of the rat. Although hypoxanthine is the
primary, if not sole, AdN degradation product within
muscle to be salvaged, we also evaluated the rates of
adenine salvage, since there is a robust activity of
APRT, relative to HPRT, in skeletal muscle (29). This
raises the potential for extracellularly derived adenine
to be incorporated into the AdN pool (5). Thus, for
completeness, we included an evaluation of both adenine and hypoxanthine as substrates for salvage. Because the fractional turnover rate of AdN is potentially
threefold higher in the high-oxidative soleus muscle
than in fast-twitch low-oxidative white sections of muscle (34), we hypothesized that significant differences in
purine salvage rates would be found among fiber types.
Furthermore, we hypothesized that purine salvage
rates would be favored over de novo synthesis and
elevated by ribose supplementation. The outcome of
this study may help explain the physiological responses of muscle after repeated days of intense exercise where AdN contents are decreased.
PURINE SALVAGE IN SKELETAL MUSCLE
233
thine, and adenine fractions were collected, and radioactivity
was counted by dual-channel liquid scintillation counting
(quench corrected to disintegrations per minute). The specific
activity of the purine base was calculated for every tissue and
perfusate sample as disintegrations per minute per nanomole. Phosphocreatine was measured using anion-exchange
HPLC as described by Wiseman et al. (40). Glycine was
separated using reverse-phase HPLC after phenylisothiocyanate derivatization of perfusate or muscle extract (34). The
glycine fraction was collected and counted by liquid scintillation counting.
To determine the muscle water content, a 150- to 250-mg
portion of gastrocnemius mixed-fiber section was dried at
60°C to a stable weight. Metabolite contents and salvage
rates are expressed at a water content of 76% (see RESULTS),
which is typical for rested rat skeletal muscle (20).
Calculations
Statistics
Analyses of variance followed by Tukey’s test were used to
detect significant differences (P ⬍ 0.05) among means. Values are means ⫾ SE.
RESULTS
Precursor Pool for Purine Salvage
Intracellular specific activity was measured, free of
the confounding influence of the perfusate radioactivity, by washing out the vascular space with perfusate
free of radioactivity and purine base. After delay for
the void volume, the [3H]adenine content (dpm) of the
venous effluent declined rapidly (Fig. 1) in a manner
that could be characterized by a double exponential
(Fig. 1, inset). The initial rate of efflux exhibited a half
time of 0.5 min, which is taken to represent washout of
the extracellular compartment (20). The second, slower
exponential (half time ⫽ 2.2 min) likely represents
[3H]adenine efflux from the intracellular space. Therefore, after a 5-min washout, we expect that essentially
all the radioactivity and purine base measured from
the muscle samples is from the intracellular space.
Accurate measurement of purine salvage rates requires knowledge and control of the precursor specific
activity. We controlled intracellular precursor specific
activity by loading the muscle with extracellular precursor. High uptake of purine base by the muscle was
established by increasing perfusate concentrations
Fig. 1. Tissue efflux of [3H]adenine after perfusate medium is
switched to zero adenine concentration containing no [3H]adenine.
Inset: 2-exponential fit to the decrease in [3H]adenine over time. Rate
constants k1 and k2 have half-time values of 0.5 and 2.2 min,
respectively.
over a 15- or 25-fold range (0.14–2.13 mM for adenine
and 0.09–2.52 mM for hypoxanthine). Muscle concentrations of adenine and hypoxanthine, measured after
perfusate washout, increased linearly (r ⫽ 0.99) with
extracellular adenine and hypoxanthine concentrations. This resulted in plantaris concentrations of
0.16–1.48 ␮mol/g for adenine and 0.07–1.66 ␮mol/g for
hypoxanthine, 3- to 100-fold higher than normal resting muscle values. More importantly, tissue adenine
and hypoxanthine specific activities increased proportionally (r ⫽ 0.99) with extracellular specific activities,
except for the lowest extracellular concentration of
adenine used (0.14 mM). Furthermore, specific activities were not different among fiber type sections. This
suggests that extracellular adenine or hypoxanthine is
the primary source for the intracellular pool in our
experiments. Therefore, we used tissue adenine and
hypoxanthine specific activities for calculation of salvage rates. Additionally, muscle samples taken at 30
min, with labeled precursor in the vasculature, yielded
specific activities similar to those at the later time
point after washout of the labeled perfusate. This suggests that extracellular and intracellular specific activities were essentially the same by 30 min. Therefore,
the specific activities measured in the 30-min muscle
samples should be valid for calculating purine salvage
rates. Furthermore, any error would overestimate the
intracellular specific activity and, therefore, underestimate the salvage rates. This is not evident in our
time-course experiments, where the first 0.5-h salvage
rates were similar to or faster than the second 0.5-h
rates (see below).
Water contents of all muscles were 76.1 ⫾ 0.17 for
the adenine animals (n ⫽ 25) and 76.1 ⫾ 0.18 for the
hypoxanthine animals (n ⫽ 16).
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Salvage rates (nmol 䡠 h⫺1 䡠 g⫺1) were calculated from the 3H
incorporated into the ATP pool (dpm/g) divided by the specific
activity of the tissue adenine or hypoxanthine (dpm/nmol)
and corrected for the time of sampling. The net amount of
label measured in the muscle AdN pool in this study likely
represents the actual rate of purine salvage, since loss of
label once incorporated into ATP should be inconsequential
(34). The turnover of the adenine ring of ATP is relatively
slow, and the specific activity of any lost adenine ring from
ATP would have a much lower specific activity (0.3–0.4%)
than the incoming precursor during salvage.
De novo synthesis rates were calculated similarly by dividing the 14C radioactivity incorporated into the ATP pool by
the specific activity of glycine from the muscle extracts (34).
234
PURINE SALVAGE IN SKELETAL MUSCLE
Effect of Time
Effect of Precursor Concentration
Figure 4 illustrates the absence of marked influence
of purine base concentration on salvage rates in the
mixed-fiber plantaris. The modest increase over adenine
concentration (⬃25%) is relatively inconsequential,
Fig. 2. Time course of hypoxanthine and adenine salvage in the
different skeletal muscle fiber types of the rat (n ⫽ 3–5/group). Note
the lack of linearity with time for adenine. Gastroc, gastrocnemius.
Fig. 3. Time course of adenine salvage in the different skeletal
muscle fiber types (n ⫽ 5) in the presence of 4.0 mM ribose. Note the
linear increase in adenine incorporation over time established by
ribose.
considering the large increase (⬃10-fold) in perfusate
concentration. Thus neither adenine nor hypoxanthine
concentration appreciably influenced salvage rates in
skeletal muscle. One exception was with the soleus
muscle, where a relatively high rate was observed at
the lowest concentration of hypoxanthine. This may be
a spurious result reflecting a greater error in determining precursor specific activity because of the small
tissue hypoxanthine content. This difference in the
soleus appears to be anomalous, since differences were
not apparent in the other fiber type sections for hypoxanthine, nor were there any differences for any of the
fiber types for adenine salvage. As a result, we have
pooled the data from the four concentrations to calculate the average salvage rate for each fiber type for
adenine and hypoxanthine.
Fig. 4. Adenine and hypoxanthine salvage rates in the plantaris
muscle as a function of increasing purine concentration ([purine]) in
the perfusate medium (n ⫽ 3–5/group).
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As shown in Fig. 2, hypoxanthine salvage (nmol/g)
increased in proportion to time despite the nearly twofold difference in rates (nmol 䡠 h⫺1 䡠 g⫺1) among fiber
types. Because the rates did not change over time, we
have averaged rates obtained from the muscles at the
30- and 65-min time points for analysis of hypoxanthine salvage rates. On the other hand, the rates of
adenine salvage decline appreciably in all fiber types
during the second 0.5 h of the experiment (Fig. 2). We
suspect that this process is specific to adenine and not
due to a general decline in tissue viability. ATP (7.61 ⫾
0.10 ␮mol/g, n ⫽ 27) contents remained high for the
length of the experiment. Furthermore, identical perfusion conditions in the hypoxanthine experiments did
not show this decrease in salvage rate. Finally, as
illustrated in Fig. 3, adenine salvage remained linear
(r ⫽ 0.99) over time through the entire experiment
with the addition of ribose. Consequently, adenine salvage is calculated using the 30-min value.
235
PURINE SALVAGE IN SKELETAL MUSCLE
Table 1. Purine salvage rates in the different skeletal muscle fiber types of the rat
Purine Salvage Rate, nmol 䡠 h⫺1 䡠 g⫺1
Precursor
n
Plantaris
Soleus
Red
White
Mean
Difference
Needed
Adenine
Hypoxanthine*
Adenine ⫹ 4.0 mM ribose‡
Hypoxanthine ⫹ 4.0 mM ribose‡
20
12
5
4
48.6 ⫾ 3.7
29.3 ⫾ 4.3
250 ⫾ 20
200 ⫾ 30
47.5 ⫾ 2.9
19.2 ⫾ 1.6†
143 ⫾ 13§
116 ⫾ 27†
55.9 ⫾ 4.2
31.8 ⫾ 2.2
262 ⫾ 25
244 ⫾ 45
35.2 ⫾ 4.6†
21.3 ⫾ 2.9†
223 ⫾ 27
148 ⫾ 14†
8.1
10.5
53
87
Gastrocnemius
Values are means ⫾ SE. Concentrations of purine base were combined in experiments without ribose; 2.0 mM purine base was used when
ribose was added. Mean difference needed to be significant, P ⬍ 0.05 by Tukey’s procedure. * Hypoxanthine salvage rates lower than adenine
salvage rates (P ⬍ 0.05). † Significantly lower than red gastrocnemius (P ⬍ 0.05). ‡ Significant ribose effect to increase purine salvage rates
(P ⬍ 0.001). § Significantly less than all other fiber sections (P ⬍ 0.05).
Muscle Fiber Type
Relationship Between Purine Salvage and
De Novo Synthesis
DISCUSSION
The present results extend the findings of Tully and
Sheehan (38), who used isolated muscle incubated in
vitro, in showing that purine salvage to AdN proceeds
at a modest rate that is not identical across skeletal
muscle fiber types. As expected from the relative activities of HPRT and APRT (29), hypoxanthine is salvaged
at a lower rate (P ⬍ 0.05) for all fiber sections than is
adenine; the greatest contrast in salvage rates between
these two purines is apparent by the exceptionally low
rate of hypoxanthine salvage in the slow-twitch red
fibers of the soleus muscle compared with the high rate
After confirming (n ⫽ 2) the reproducibility of de
novo synthesis rates that we measured previously (n ⫽
4) (34, 35), we examined de novo synthesis in the
presence of adenine. As illustrated in Fig. 6, de novo
synthesis was significantly reduced (P ⬍ 0.005) in all
Fig. 5. Increase in adenine salvage in the plantaris muscle as a
function of ribose concentration ([ribose]) in the perfusion medium
(n ⫽ 2–5/group). Inset: same results expressed on a logarithmic scale
for ribose.
Fig. 6. Influence of adenine and ribose on de novo synthesis rates in
soleus, red gastrocnemius, and white gastrocnemius muscle sections.
Addition of adenine (0.2, 0.5, or 1.0 mM, n ⫽ 5 combined) to the
perfusion medium decreases de novo synthesis rates with (P ⬍ 0.05)
and without (P ⬍ 0.005) ribose (4.0 mM ribose and 0.2 mM adenine,
n ⫽ 2) stimulation. Inherent de novo synthesis rates were taken from
our previous work (34). Ribose-enhanced rates (n ⫽ 6) include data
from our previous work (n ⫽ 4) (35).
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Purine salvage rates differ modestly, but significantly, among skeletal muscle fiber types (Table 1).
The low-oxidative white gastrocnemius generally has
the lowest salvage rate, whereas the high-oxidative red
gastrocnemius exhibits the fastest. Interestingly, hypoxanthine salvage rates in the high-oxidative slowtwitch soleus were similar to those in the low-oxidative
fast-twitch white gastrocnemius. Salvage rates of adenine are nearly twice as high as those of hypoxanthine.
Moreover, the addition of 4.0 mM ribose increased
adenine salvage three- to sixfold and hypoxanthine
salvage six- to eightfold among fiber types. As illustrated for the plantaris muscle in Fig. 5, this influence
of ribose is concentration dependent.
fiber types by the addition of adenine [0.2 mM (n ⫽ 2),
0.5 mM (n ⫽ 1), or 1.0 mM (n ⫽ 2)]. Similarly, perfusion
with adenine markedly reduced (P ⬍ 0.05) the increase
in de novo synthesis rates stimulated by 4.0 mM ribose
supplementation (n ⫽ 2).
236
PURINE SALVAGE IN SKELETAL MUSCLE
contributor to the total purine salvage rates measured
in our study.
The relatively stable rates of purine salvage observed over a ⬃15- to 25-fold range of precursor supply
(Fig. 4) imply that a low purine supply, possibly characteristic of that found physiologically, is sufficient to
sustain the modest rates of purine salvage to AdN
normally found in skeletal muscle. This is similar to
the findings of Brown et al. (7) for mature cardiac
myocytes incubated in culture. Thus, although high
concentrations of purine precursor were used experimentally in this study, they are not needed physiologically. It is well established that PRPP and its precursor ribose-5-phosphate are rate limiting in AdN
synthesis for de novo synthesis (6) and purine salvage
pathways (23). This is why AdN synthesis is markedly
accelerated by provision of glucose (6) and ribose (42,
43). We have verified that ribose supply increases adenine and hypoxanthine salvage rates by three- to
eightfold (Table 1). Furthermore, we have shown that
the influence of ribose appears to be saturable at concentrations somewhat higher than the 10 mM maximum used. Interestingly, perfusing the muscle with
4.0 mM ribose not only increased the adenine salvage
rate dramatically but also removed the nonlinearity in
adenine salvage that was observed over time. The
decline in adenine salvage during the second 0.5 h of
perfusion observed in the absence of ribose (Fig. 2) was
eliminated by 4.0 mM ribose. This suggests that, in the
case of adenine, but not hypoxanthine, ribose-5-phosphate became limiting over time. The nonlinearity is
not likely due to the characteristic impediment to purine salvage caused by the absence of glucose (6), since
5 mM glucose was maintained in the perfusion medium. On the other hand, the higher inherent rates of
adenine salvage, compared with hypoxanthine (Table
1), are consistent with PRPP being limiting. However,
direct measurements of muscle ribose-5-phosphate and
PRPP contents will be needed to test this possibility.
Our results illustrate the reciprocity between the de
novo and purine salvage pathways, likely through competition for PRPP (41). The large decline that was
observed in the rates of de novo synthesis on the
addition of adenine to the perfusion medium (Fig. 6)
can be attributed to the greater activities and affinities
of two key enzymes in the salvage pathway (APRT and
HPRT) for PRPP than of amidotransferase, the ratelimiting step in the de novo pathway (27, 39). Interestingly, the higher the inherent rate of de novo synthesis
among fiber sections, the greater was the reduction
with adenine. This is consistent with competition for
available PRPP. Thus the relatively low rate of de novo
synthesis, characteristic of the low-oxidative white fibers, was reduced the least by adenine. We also have
shown that the accelerated rate of de novo synthesis,
established by ribose supplementation, could not be
sustained during conditions of accelerated adenine salvage. De novo synthesis rates declined; however, the
absolute rates of de novo synthesis in the presence of
adenine ⫹ ribose were better preserved than in the
absence of ribose (Fig. 5). This again places importance
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of adenine salvage for this same fiber section (Table 1).
Results of the present study were obtained using an
isolated perfused hindlimb preparation, where the purine substrate was delivered through the vasculature
at a high flow rate over a wide range of concentrations.
Even though the uptake of purines by myocytes in
culture can be achieved at low concentrations of purines [e.g., Michaelis-Menten constant of 1–10 ␮M (7)],
we used relatively high purine concentrations to control the precursor pool specific activity and to assess
the influence of supply concentration in the complex
whole muscle tissue. After extensive washout of the
extracellular compartment (Fig. 1), tissue specific activities of adenine and hypoxanthine were found to be
similar to the extracellular perfusate specific activities
over a broad range of plasma concentrations. This
provides assurance that our measured rates of purine
salvage have not been confounded by a mixture of
intracellular- and extracellular-derived precursor.
Thus we believe that our results represent valid rates
of purine salvage.
Another potential complication in the interpretation
of our results stems from purine metabolism in nonskeletal muscle cells within the tissue. Because the
salvage precursor obligatorily passes through the vasculature to reach the myocytes, considerable metabolism by the vascular endothelium could act as a barrier
and impact salvage measurements. Indeed, cultured
endothelial cells are capable of purine salvage and de
novo synthesis (9). Although skeletal muscle endothelial cells have been implicated in transport and metabolism at micromolar concentrations of purines in perfusion studies (12), we suspect that purine metabolism
by the endothelium would become quantitatively less
important at the higher precursor concentrations used
here. Purine base concentrations in the present study
were 3- to 100-fold higher than normally found in
whole muscle tissue. Thus it seems unreasonable to
assert that uptake of adenine and hypoxanthine was
limited to the endothelium, which represents an exceedingly small fraction (⬃0.5%) of the total tissue
volume. Furthermore, salvage rates in the endothelial
cells would have to be exceptionally high to account for
a substantial fraction of the response of the whole
muscle tissue. For example, capillary endothelial cells
would have to exhibit a salvage rate ⬃20-fold greater
than that of the myocytes to contribute ⬃10% of the
purine incorporated into AdN in this study. This estimate is calculated on the measured capillary surface
area of 25 mm2/mm3 fiber for mixed-fiber muscle (28),
an in vivo cell height of 0.2 ␮m (31), and an extracellular volume of 15% (20). Additionally, the soleus muscle, which has an exceptionally high capillary density
and, consequently, a high content of endothelium compared with the other fiber sections examined, has one
of the lowest hypoxanthine salvage rates. If the endothelium or other component of the vasculature were a
major site of purine salvage, the soleus would be expected to have a relatively high salvage rate. As a
result, it is unlikely that the endothelium is a major
PURINE SALVAGE IN SKELETAL MUSCLE
The influence of ribose may be particularly germane in
this context. The large increases in purine salvage with
ribose in the perfusion medium suggest that ribose
supplementation could improve ATP recovery during
repeated days of intense cycle exercise. Future experiments are needed to test this hypothesis.
In conclusion, we show that purine salvage rates to
AdN are greater for adenine than for hypoxanthine
and differ among the various skeletal muscle fiber
types. The marked increase in purine salvage that
occurs with ribose supply could be important during
conditions where purine salvage is critical to the maintenance of the AdN pool within the muscle.
This study was supported by National Institute of Arthritis and
Musculoskeletal and Skin Diseases Grant AR-21617.
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