INTRODUCTION

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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 · h¹ · g muscle¹, 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 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.

	MATERIALS AND METHODS

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Animals

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 µU/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 administered 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 · min¹ · g perfused tissue mass¹ (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 µCi/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.

Analyses

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, hypoxanthine, 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

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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).

Statistics

	RESULTS

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Precursor Pool for Purine Salvage

View larger version (22K): [in this window] [in a new window]   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.

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 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 µmol/g for adenine and 0.07-1.66 µmol/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).

Effect of Time

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View larger version (17K): [in this window] [in a new window]   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.

View larger version (21K): [in this window] [in a new window]   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.

Effect of Precursor Concentration

View larger version (17K): [in this window] [in a new window]   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).

Muscle Fiber Type

View larger version (19K): [in this window] [in a new window]   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.

Relationship Between Purine Salvage and De Novo Synthesis

View larger version (34K): [in this window] [in a new window]   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).

	DISCUSSION

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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 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 µM (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 µm (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 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 rate-limiting 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 on PRPP availability, while the relative capacity for de novo synthesis is secondary to the more efficient salvage pathway for AdN synthesis. This does not mean that the de novo pathway is inconsequential, as illustrated by knockout mice that lack the HPRT gene (1, 21) or lack the HPRT and APRT genes (11). Neither knockout mouse is an adequate behavioral model for their intended purpose to assess the pathogenesis of Lesch-Nyhan syndrome, but both appear healthy and have proven useful in the study of purine management. The elevated rates of de novo synthesis measured in some tissues of the HPRT mutant mice (1) likely contribute to the apparent health of the animal. It is likely that de novo synthesis is also elevated in the double-knockout animals. Even though these animals appear to function normally at rest, analysis of purine management during or after intense exercise, where AdN degradation is accelerated, may elicit additional differences in AdN metabolism.

The need for de novo synthesis and purine salvage of AdN is probably most acute after circumstances that cause exaggerated AdN degradation, such as ischemia and/or intense muscle contractions. De novo synthesis of AdN proceeds at a relatively low rate in skeletal muscle (34), is reduced during muscle contractions (35), and is tempered in deference to purine salvage (Fig. 5) (30, 41). Thus the process of purine salvage must be important in skeletal muscle, especially after intense exercise. AdN degradation in humans, as evident by release of hypoxanthine, continues for an extended time during recovery (18) in a manner related to exercise intensity (33). This degradation can be extensive, with as much as 40-50% of the muscle decline in ATP accounted for by efflux of hypoxanthine (15). Any net efflux of inosine and/or hypoxanthine from the muscle requires compensation, if the muscle's AdN pool is to be maintained. Interestingly, a number of studies have shown that muscle ATP concentration is significantly lower (15-20%) after several days of intense cycle exercise (16, 19, 32). Either the muscle is remodeling to a phenotype exhibiting a lower ATP concentration or AdN production processes are inadequate to fully recover muscle ATP concentration. An increase in the enzymatic activity of HPRT in high-intensity trained individuals (17) implies that adaptations have occurred to enhance ATP recovery. Thus it may be that an insufficiently low rate of purine salvage is the cause for the reduced ATP concentration after repeated days of high-intensity cycle exercise. Results of the present study support this hypothesis in showing that the rates of hypoxanthine salvage are lowest in the low-oxidative fast-twitch white fibers. Furthermore, this fiber type exhibits the lowest rate of de novo synthesis (34). Although there is not a precise equivalent of this phenotype in human muscle, the fast-twitch fibers of humans are the most likely to produce copious amounts of IMP during intense exercise that can lead to AdN degradation (10, 22). If our findings in rat muscle are at all applicable to human muscle, then the decline in muscle ATP after repeated days of intense cycling could be due to insufficient resynthesis of AdN. 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.