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. 1999 Aug 1;518(Pt 3):761–768. doi: 10.1111/j.1469-7793.1999.0761p.x

The effect of muscle contraction on the regulation of adenosine formation in rat skeletal muscle cells

Ylva Hellsten 1
PMCID: PMC2269451  PMID: 10420012

Abstract

  1. The present study examined the effect of muscle contraction on the rate of extracellular adenosine formation and on the distribution of 5′ nucleotidase in primary rat skeletal muscle cells in culture. Experiments were also performed to determine whether the muscle cells release a metabolite upon contraction which may influence the extracellular production of adenosine.

  2. Muscle contraction, induced by electrical stimulation, increased (P < 0.05) the rate of adenosine formation in the presence of physiological concentrations (2 and 5 μM) of adenosine monophosphate (AMP). Muscle contraction also led to an increase (P < 0.05) in the maximal rate of extracellular adenosine formation from 4.09 ± 0.19 to 7.04 ± 0.27 μmol (g protein)−1 min−1. Similarly, homogenates of contracted muscle cells had a higher (by 19.5 ± 10.5 %; P < 0.05) AMP 5′ nucleotidase activity than homogenates of control cells.

  3. Addition of buffer from contracted cells to control cells induced an elevation (18.4 ± 5.3 %; P < 0.05) in the rate of adenosine formation. The rate of adenosine formation was also increased with decreased intracellular adenylate charge (P < 0.05).

  4. Cell homogenates treated with detergent had a higher (by 58.0 ± 16.3 %; P < 0.05) AMP 5′ nucleotidase activity than untreated homogenates, suggesting the existence of an enclosed pool of 5′ nucleotidase within the muscle cells. The rate of adenosine formation in the detergent-treated homogenates was similar for electrically stimulated and non-electrically stimulated cells.

  5. The present data show that muscle contraction induces an enhanced extracellular adenosine production via an increase in the activity of ecto AMP 5′ nucleotidase. The activity of 5′ nucleotidase can be elevated via a compound released by muscle cells during contraction and by alteration in intracellular adenylate charge. It is furthermore proposed that the extracellular adenosine formation is increased by translocation of 5′ nucleotidase from an enclosed intracellular pool to the muscle membrane.

In skeletal muscle adenosine is thought to be of importance for many aspects, such as for the regulation of blood flow and for glucose uptake (Dobson et al. 1971; Espinal et al. 1983; Vergauwen et al. 1994; Han et al. 1998), events that may be altered during muscle contraction. In several studies adenosine has been found to be released from contracting animal muscle (e.g. Dobson et al. 1971; Ballard, 1991) and the concentration of adenosine in the interstitium of exercising human muscle has been found to increase with increasing work intensity (Hellsten et al. 1998).

There are two major sources of adenosine in tissues, non-specific phosphatases and adenosine monophosphate (AMP) 5′ nucleotidase (EC 3.1.3.5), of which the latter probably is the most important in skeletal muscle. In most cell types studied 5′ nucleotidase, which hydrolyses AMP to adenosine and inosine monophosphate (IMP) to inosine, exists both as a soluble cytosolic enzyme and as an ecto enzyme (Newby et al. 1975) bound to the membrane via an inositol phosphoglycan (Stochaj et al. 1989; Misumi et al. 1990). The kinetics, substrate specificity and characteristics of the enzyme seem to differ between the soluble and the membrane-bound form and also between tissues (Zimmerman, 1992). We have observed in a previous study that the extracellular adenosine formation is enhanced in electrically stimulated rat skeletal muscle cells in culture and have attributed this observation to an enhanced adenosine formation via the membrane-bound (ecto) form of AMP 5′ nucleotidase (Hellsten & Frandsen, 1997). It remains unclear, however, how the rate of extracellular adenosine formation may be regulated by muscle contraction. One possibility is that the activity of AMP 5′ nucleotidase in the muscle membrane may be enhanced by translocation of the enzyme from an intracellular fraction. Such a movement of 5′ nucleotidase has previously been described for other cell types (Stanley et al. 1980). Another possibility is that cells in the muscle tissue release a metabolite upon muscle contraction that acts as a positive effector for AMP 5′ nucleotidase, thereby increasing the rate of adenosine formation. Several metabolites, such as lactate, nitric oxide, potassium and ATP are known to increase in the extracellular fluid in contracting skeletal muscle (Frandsen et al. 1998; Hellsten et al. 1998; Maclean et al. 1999). Similar to adenosine these substances are vasoactive and have been shown to interact with adenosine (Newby et al. 1975; Ballard 1991; Marshall et al. 1993; Minamino et al. 1997). Finally, it is possible that 5′ nucleotidase, similar to a number of muscle enzymes, may be modified by an increase in intracellular calcium which occurs upon muscle contraction.

Thus, the aims of the present study were to examine (a) how muscle contraction changes the kinetics of adenosine formation, (b) whether a change in distribution of AMP 5′ nucleotidase occurs upon stimulation and (c) whether the extracellular production of adenosine may be influenced by an increase in extracellular potassium, nitric oxide, lactate or ATP, or by an altered intracellular calcium level. The enzyme 5′ nucleotidase was especially focused on including an evaluation of the substrate specificity of the enzyme. Skeletal muscle cells in culture were utilized as the experimental model in order to exclude adenosine formation in vascular cells.

METHODS

Materials

Hepes, EHNA (erythro-9-(2-hydroxy-3-nonyl) adenine), Triton X-100, adenosine 5′ triphosphate (ATP), adenosine 5′ diphosphate (ADP), adenosine 5′ monophosphate (AMP), inosine monophosphate (IMP), adenosine, inosine, hypoxanthine, potassium cyanide (KCN), β-glycerophosphate and DNAse were obtained from Sigma. Lactate was obtained from Boehringer Mannheim GmbH. Terbutalin (Bricanyl; 0.5 mg ml−1) was obtained from Draco, Lund, Sweden. Dulbecco's modified Eagle's medium (DMEM), fetal calf serum, horse serum and trypsin were purchased from Gibco.

Isolation and culture of rat primary skeletal muscle cells

All experiments complied with the European Convention for the Protection of Vertebrate Animals Used for Experiments or Other Scientific Purposes (Council of Europe no. 123, Strasbourg, France, 1985). Rats (21 days pregnant) were killed with carbon dioxide released by the addition of water to 1 kg of dry-ice, as well as carbon dioxide injected from a tank into an air-tight container of approximately 5 l volume, a procedure that induced death in less than 3 min. The hind limbs of the 21-day-old rat embryos were then used to provide cell cultures (Daniels, 1990). In brief, muscle tissue was dissected out from hind limbs under a microscope and digested with 0.2 % trypsin and 0.01 % DNAse at 37°C for 30 min. DMEM containing 10 % fetal calf serum and 10 % horse serum (growth medium) was added to the suspension and the remaining clumps were dissociated by trituration with a 10 ml pipette. The cell suspension was centrifuged at 300 g for 8 min and the supernatant was discarded. Twenty millilitres of growth medium was added and the suspension was again triturated with a 10 ml wide-bore pipette to dissociate aggregated cells. The suspension was filtered through a 70 μm nylon mesh and the cells were seeded out onto two 90 mm Petri dishes for 45 min during which time fibroblasts attached to the bottom of the dish whereas the myoblasts remained in suspension. The medium was removed from the dishes and the dishes were discarded. Cells were counted and seeded out onto 90 mm dishes coated with 0.1 % gelatine. For the purpose of further purification of the myocytes, fibroblasts and other contaminating cell types were removed by dispase treatment 48 h after seeding as described by Daniels (1990). All experiments were performed on first passage cells 11-12 days after dispase re-plating.

Effect of muscle contraction on the rate of adenosine formation with physiological concentrations of AMP

The effect of muscle contraction on the rate of adenosine formation for low extracellular concentrations of AMP in the physiological range (i.e. 1-5 μM; Hellsten et al. 1998) was examined in intact non-stimulated and stimulated muscle cells (n = 6). AMP concentrations of 2 and 5 μM were used. Dishes with muscle cells were rinsed with Krebs-Ringer buffer containing (mM): 118.5 NaCl, 24.6 NaHCO3, 23.8 KCl, 1.18 MgSO4.7H2O, 0.71 KH2PO4.3H2O, 3.36 CaCl.2H2O, 25 bicarbonate and 25 Hepes; pH 7.4. EHNA was added to the medium to a final concentration of 5 μM to prevent adenosine deamination (Jacobs et al. 1988). Higher concentrations of EHNA did not reduce the activity of adenosine deaminase further. Krebs-Ringer buffer (1000 μl) was then added to each dish. The reaction was carried out at +30°C and started with the addition of either 2 or 5 μM AMP to the buffer. Samples of buffer for the determination of adenosine were collected from the dishes after 2, 5 and 10 min and frozen at -80°C until time of analysis. After completion of the experiment the cell dishes were rinsed with PBS, and the muscle cells were scraped off with a rubber policeman and saved for protein determination. The rate of adenosine formation was linear during the 10 min of measurements and the rate of adenosine formation was calculated as the amount of adenosine formed in the buffer between sampling times, divided by the time between sampling and divided by the total amount of cell protein in the dish. Values are expressed in μmol (g protein)−1 min−1.

Experiments were conducted pairwise with and without electrical stimulation. For the measurements of adenosine formation during muscle contraction electrical stimulation was applied to the cells from immediately prior to the addition of AMP and throughout the experiment. The muscle cells were stimulated via silver chloride electrodes placed at the inside edges of the dishes. The stimulation protocol consisted of 0.7 s trains with a 0.3 s pause between trains, the trains consisting of stimuli of 1 ms pulse width, 0.01 s pulse interval delivered at 50 V.

Kinetics of extracellular adenosine formation

The kinetics of adenosine formation was examined in intact non-stimulated and in electrically stimulated muscle cells (n = 7). The reaction was carried out at +30°C and started with the addition of 2, 5, 10, 50, 100, 250, 500 or 2000 μM AMP to the buffer. A new cell dish from the same cell batch was used to determine the rate of adenosine formation for each separate AMP concentration. Experiments were conducted pairwise with and without electrical stimulation and in series with the different concentrations of AMP. The experimental procedure, stimulation protocol and calculations were otherwise exactly as that described under ‘Effect of muscle contraction on the rate of adenosine formation with physiological concentrations of AMP’.

The rate of adenosine formation was linear during the 10 min of measurements and calculated as the adenosine concentration in the buffer formed between sampling times, divided by the time between sampling and divided by the total amount of protein in the cell dish. Values are expressed in μmol (g protein)−1 min−1. The data were used to calculate Vmax and Km values for adenosine formation, with and without electrical stimulation, with the Michaelis-Menten equation.

For the measurements of kinetics of adenosine formation during muscle contraction electrical stimulation was applied to the cells from immediately prior to the addition of AMP and throughout the experiment.

Adenosine formation in skeletal muscle cell homogenates

The maximal rate of adenosine formation was determined in homogenates of non-stimulated muscle cells (n = 5) and of muscle cells that immediately prior to homogenization had been electrically stimulated for 10 min, according to the protocol described above (n = 5). The procedure was as follows: dishes with cells were placed on ice, rinsed with ice-cold Krebs-Ringer buffer containing 25 mM bicarbonate, 25 mM Hepes, 5 μM EHNA, pH 7.4, and the cells were rapidly scraped off and transferred to a chilled Potter-Elvehjem glass homogenizer. Krebs-Ringer buffer (1200 μl) was added to approximately 1.5 mg of cells, and the cells were homogenized manually whilst on ice. The homogenate was divided into two parts where one part was further homogenized with the addition of 2 % Triton X-100, in order to examine if there was a detergent-soluble enclosed pool of 5′ nucleotidase. The measurements of adenosine formation were performed at +30°C and the reaction was started by the addition of AMP to a final concentration of 2 mM. Samples of the homogenate for the determination of adenosine were collected at 2, 5 and 10 min. An aliquot of the homogenate was saved for protein determination. The rate of adenosine formation was linear during the 10 min. The rate of adenosine formation was calculated as the amount of adenosine formed, divided by time and by the total amount of protein in the homogenate and are expressed in μmol (g protein)−1 min−1.

Fractionation of cell homogenate

The rate of adenosine formation was determined in the crude and cytosolic fraction in non-stimulated and stimulated cells (n = 6). Cells were manually homogenized in Krebs-Ringer buffer as described above under ‘Adenosine formation in skeletal muscle cell homogenates’. The homogenate was centrifuged at 100 000 g at +4°C for 120 min. The supernatant was collected and the pellet was resuspended in Krebs-Ringer buffer. The rate of adenosine formation in the supernatant and in the resuspended pellet was measured as for whole homogenates.

Contribution of 5′ nucleotidase to adenosine formation

The contribution of 5′ nucleotidase versus non-specific phosphatases to the total rate of adenosine formation was determined in intact non-stimulated and electrically stimulated (n = 10) cells and in homogenates of non-stimulated and electrically stimulated (n = 12) cells. These experiments were performed as described above under ‘Kinetics of extracellular adenosine formation’ and ‘Adenosine formation in skeletal muscle cell homogenates’ except that the non-specific phosphatase activity was inhibited by the addition of 100 mM β-glycerophosphate to the reaction buffer (Newby et al. 1975). For measurements in cell homogenates, the inhibitor was also used at the same concentration in the homogenate. Higher concentrations of β-glycerophosphate did not further reduce the formation of adenosine in the presence of AMP. The experiments were conducted pairwise with and without the addition of β-glycerophosphate added to the intact cultures or the cell homogenates.

Activity of 5′ nucleotidase with IMP as substrate

The activity of 5′ nucleotidase with IMP as substrate was determined in intact non-stimulated and electrically stimulated cells as well as in muscle homogenates to examine whether the relative activity of the enzyme with AMP versus IMP as substrate was similar in the various fractions of the muscle cells (n = 6) and whether the activity (n = 8) with IMP as substrate was altered with muscle contraction. For the measurements of 5′ nucleotidase activities with IMP as substrate, the same experimental procedures and calculations as described under ‘Effect of muscle contraction on the rate of adenosine formation with physiological concentrations of AMP’ and ‘Adenosine formation in skeletal muscle cell homogenates’ were used, except that the reaction was started by the addition of IMP to a final concentration of 2 mM. Buffer samples obtained from the cell dishes were analysed for inosine and the activity of 5′ nucleotidase is presented as μmol (g protein)−1 min−1.

Transfer of buffer from stimulated to non-stimulated cells

The following experiment was conducted to examine whether the increased rate of adenosine formation that occurred upon contraction was due to an effector released from the muscle cells. A cell dish was rinsed with Krebs-Ringer buffer and 1200 μl of Krebs-Ringer buffer was then added. The dish was electrically stimulated for 10 min according to the protocol described above. Upon termination of stimulation 1000 μl of the buffer was removed from the stimulated cells and transferred to another dish of rinsed, non-stimulated cells. These non-stimulated cells were incubated with the buffer for 10 min. The rate of extracellular adenosine formation was then measured during 10 min at +30°C after the addition of AMP to a final concentration of 2 mM. Buffer samples were collected at 2, 5 and 10 min for the determination of adenosine. Cell protein was determined for each dish after the experiment. The rate of adenosine formation of these cells was compared with that of matched non-stimulated control cells that were incubated in Krebs-Ringer buffer for 10 min prior to measurements.

The effect of potassium, lactate, sodium nitroprusside, ATP, iodoacetic acid and terbutalin on the rate of extracellular adenosine formation

A series of experiments was performed to assess whether the positive effect of transferring buffer from stimulated to non-stimulated cells on the rate of adenosine formation was due to the effect of a modulating substance released from the muscle cells. In addition the effect of iodoacetic acid, which induces a lowering of the intracellular adenylate charge, and the effect of terbutaline, which increases the intracellular calcium level, were examined. The compounds used were: KCl (10 mM), lactate (2.5 and 5 mM), sodium nitroprusside (a nitric oxide donor; 10 μM), ATP (50 μM), iodoacetic acid (0.5 mM) and terbutaline (5 μM).

Cell dishes were rinsed with Krebs-Ringer buffer and 1000 μl of buffer was added to each dish. The compound to be tested was added to a dish for 10 min. The reaction was started by the addition of AMP to a final concentration of 2 mM, and carried out for 10 min at +30°C. The experiments were conducted pairwise with matched, untreated controls. Procedures and calculation of the rate of adenosine formation were otherwise as described under ‘Effect of muscle contraction on the rate of adenosine formation with physiological concentrations of AMP’.

Effect of electrical stimulation on the intracellular adenylate charge

The intracellular adenylate charge was assessed in non-stimulated and electrically stimulated skeletal muscle cells (n = 5). Muscle cells, either incubated with Krebs-Ringer buffer for 10 min or electrically stimulated for 10 min (as described under ‘Effect of muscle contraction on the rate of adenosine formation with physiological concentrations of AMP’), were rapidly placed on ice and rinsed with ice-cold buffer. The cells were extracted for 20 min on ice with 1 M perchloric acid (500 μl). Adenylate charge was determined as the mole fraction of ATP plus half the mole fraction of ADP divided by the total adenylate pool (Atkinson, 1968).

Analysis

Muscle and media nucleotide, nucleoside and nucleobase concentrations were determined by reverse phase HPLC as previously described (Tullson et al. 1990). Cell protein was determined with BCA protein assay (Pierce, Rockford, IL, USA).

Statistics

Data are presented as means ±s.e.m. Medians were compared using Wilcoxon's non-parametric test. Significance was established at P < 0.05. n equals the number of rat cell batches, i.e. the number of rats.

RESULTS

Effect of muscle contraction on the rate of adenosine formation

The effect of contraction on the rate of adenosine formation for low, physiological concentrations of AMP was examined. The rates of adenosine formation were found to increase from 0.07 ± 0.01 to 0.10 ± 0.02 μmol (g protein)−1 min−1 with 2 μmol AMP (n = 6; P < 0.05) and from 0.15 ± 0.06 to 0.23 ± 0.07 μmol (g protein)−1 min−1 with 5 μmol AMP (n = 6; P < 0.05).

The kinetics of adenosine formation were assessed and the maximal rate of extracellular adenosine formation in intact non-stimulated skeletal muscle cells was determined as 4.09 ± 0.19 μmol (g protein)−1 min−1 (n = 7; Fig. 1). Of this total adenosine formation 84.3 ± 3.5 % could be explained by the activity of ecto AMP 5′ nucleotidase as assessed by inhibition of non-specific phosphatases (n = 10). Electrically stimulated muscle cells had a greater maximal rate of extracellular adenosine formation (7.04 ± 0.27 μmol (g protein)−1 min−1) than non-stimulated cells (P < 0.05, n = 7; Fig. 1). The contribution of ecto AMP 5′ nucleotidase to the maximal rate of extracellular adenosine formation during electrical stimulation (86.6 ± 6.4 %) was not different from that of non-stimulated cells (P >0.05; n = 6). In accordance with this, the contraction-induced increase in the rate of extracellular adenosine formation was not different with and without inhibition of non-specific phosphatases (P > 0.05), indicating that the effect of contraction was due to an increase in the activity of ecto AMP 5′ nucleotidase. The Km value for the stimulated cells was higher (206 ± 24.5 μM; P < 0.05; n = 7) than that of the non-stimulated cells (93.5 ± 15.9 μM; n = 7; Fig. 1).

Figure 1. Kinetics of extracellular adenosine formation in non-stimulated and electrically stimulated skeletal muscle cells in culture.

Figure 1

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The rate of extracellular adenosine formation (v) was measured in cultures of non-stimulated skeletal muscle cells (^) and during electrical stimulation of skeletal muscle cells (n = 7; •) at concentrations from 2 to 2000 μM in the medium. The curve was constructed from the Michaelis-Menten equation. The calculated values for Vmax and Km were both significantly greater (P < 0.05) for the electrically stimulated cells than for the non-stimulated cells.

Adenosine formation in whole skeletal muscle cell homogenates and fractions of this

In whole homogenates of muscle cells the activity of 5′ nucleotidase activity was higher in the electrically stimulated (19.5 ± 10.5 %; P < 0.05; n = 5) than in the non-stimulated cells. The contribution of AMP 5′ nucleotidase to the adenosine formation amounted to 85.7 ± 2.3 and 84.2 ± 2.3 % for the non-stimulated and the stimulated cells, respectively (n = 12). In homogenates that had been homogenized with the addition of Triton X-100 the AMP 5′ nucleotidase activity was significantly higher, 58.0 ± 16.3 and 40.9 ± 11.4 %, for the non-stimulated and the stimulated cells, respectively, than in homogenates without addition of Triton X-100 (P < 0.01; n = 5; Fig. 2). In homogenates treated with Triton X-100 there was no significant difference in AMP 5′ nucleotidase activity between the non-stimulated and the electrically stimulated cells (n = 5; P >0.05; Fig. 2).

Figure 2. Activity of AMP 5′ nucleotidase in untreated and Triton X-100-treated homogenates of non-stimulated and electrically stimulated skeletal muscle cells in culture.

Figure 2

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Activity of AMP 5′ nucleotidase (v) in untreated (Hom) and Triton X-100-treated (Hom + TX-100) homogenates of non-stimulated (n = 5; ▪) and electrically stimulated (n = 5; Inline graphic) rat skeletal muscle cells. The activities were assessed by the addition of AMP to a final concentration of 2000 μM in the homogenate. * denotes P < 0.05; ** denotes P < 0.01 significant difference between non-stimulated and stimulated cells.

With the assumption that the detergent treatment made all of the 5′ nucleotidase activity available, it was estimated that the ecto form of muscle AMP 5′ nucleotidase constituted approximately 48 and 55 % of the total activity of muscle 5′ nucleotidase in non-stimulated and stimulated muscle cells, respectively.

Measurements of the activity of the nucleotidase present in the particulate versus the cytosolic fraction (100 000 g) of the muscle cells showed that the activity in the cytosolic fraction could account for approximately 17.8 ± 1.8 and 18.2 ± 0.8 % of the total activity in non-stimulated and stimulated cells, respectively (n = 6).

Activity of 5′ nucleotidase with IMP as substrate

The activity of ecto 5′ nucleotidase with IMP as substrate, measured in intact cells, increased from 1.44 ± 0.29 to 2.73 ± 0.52 μmol (g protein)−1 min−1 (P < 0.05; n = 8) with electrical stimulation. The activity of 5′ nucleotidase with IMP as substrate was higher (68.9 ± 10.9 %; P < 0.05; n = 8; Fig. 3) in homogenates treated with Triton X-100 compared with untreated homogenates, suggesting the presence of an enclosed intracellular pool of IMP 5′ nucleotidase. The proportion of the IMP ecto 5′ nucleotidase activity (55 %) compared with the total activity (ecto form + soluble form + enclosed intracellular form) was similar to that estimated with AMP as substrate. The activity of 5′ nucleotidase with IMP as substrate relative to the activity with AMP as substrate was similar in non-treated homogenates and Triton X-100-treated homogenates (n = 6), indicating a similar substrate specificity for the enclosed pool of the enzyme as for the ecto and cytosolic form.

Figure 3. Distribution of IMP 5′ nucleotidase activity in cultured skeletal muscle cells.

Figure 3

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Activities of IMP 5′ nucleotidase in intact skeletal muscle cells (n = 8; Ecto), untreated muscle cell homogenates (n = 8; Hom) and muscle cell homogenates treated with Triton X-100 (Hom + TX-100). The activities were determined by the addition of IMP to a final concentration of 2000 μM and the data are expressed as a percentage of the IMP 5′ nucleotidase activity in non-detergent-treated muscle cell homogenates. * denotes P < 0.05.

The effect of potassium, lactate, sodium nitroprusside, terbutaline, KCN, ATP and altered adenylate charge on the formation of adenosine

Non-stimulated cells that received buffer from cells that had been electrically stimulated had a greater (18.4 ± 5.3 %) rate of extracellular adenosine formation compared with that of non-stimulated cells (4.92 ± 0.78 vs. 4.11 ± 0.62 nmol (g protein)−1 min−1; P < 0.01; n = 8).

Addition of potassium, lactate, sodium nitroprusside, terbutaline, or ATP had no significant effect on the rate of extracellular adenosine formation. Iodoacetic acid, which altered the intracellular adenylate charge from 0.86 ± 0.09 to 0.41 ± 0.02 (P < 0.05; n = 7; Table 1), induced a significant alteration in the extracellular formation rate of adenosine (P < 0.05; Table 1).

Table 1. The effect of compounds released upon muscle contraction, and of iodoacetic acid and terbutalin on the rate of extracellular adenosine formation in cultured skeletal muscle cells.

Compound Rate of adenosine formation (% of control)
Lactate (2.5 mM) 91.8 ± 16.4 (n = 5)
Lactate (5 mM) 94.4 ± 11.2 (n = 6)
Potassium (10 mM) 95.6 ± 15.0 (n = 7)
Sodium nitroprusside (10 μM) 130.5 ± 19.5 (n = 7)
ATP (50 μM) 104 ± 13 (n = 4)
Iodoacetic acida (0.5 mM) 120.4 ± 8.1 (n = 7; P < 0.05)
Terbutalineb (5 μM) 108.5 ± 11.6 (n = 8)
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The muscle cells were incubated with the respective compound for 10 min prior to measurement of adenosine formation. The activity is expressed as a percentage of non-treated matched control cells.

a

Added to induce a lowered intracellular adenylate charge.

b

Added to induce an increased intracellular calcium level.

The intracellular (n = 5) adenylate charge values were similar in control cells and in cells stimulated electrically for 10 min (0.92 ± 0.01 vs. 89.4 ± 0.01).

DISCUSSION

The present data show that muscle contraction enhances the rate of extracellular adenosine formation in the presence of both physiological and saturating concentrations of AMP. The contraction-induced increase in adenosine formation was unaffected by inhibition of non-specific phosphatases and could thus be attributed to an increase in the activity of ecto AMP 5′ nucleotidase. The contribution of intracellular adenosine to the extracellular concentration could only have been negligible since AMP was added extracellularly and was thus not available for the intracellular enzyme. Furthermore, without the addition of AMP, the total contraction-induced extracellular accumulation of adenosine is in the order of 15 nmol (g protein)−1 min−1 (< 0.2 %). The calculated Km value was 94 μM for non-stimulated cells and 206 μM for the stimulated cells. These Km values lie well above observed levels for interstitial AMP concentrations of human muscle (67 nM; Hellsten et al. 1998), and estimated interstitial AMP levels of perfused canine hearts (100-220 nM; Kroll & Stepp, 1996). Together, these findings suggest that the rate of extracellular adenosine formation in skeletal muscle in vivo is determined both by the concentration of AMP in the interstitium and by the activity of ecto AMP 5′ nucleotidase.

Homogenates of non-stimulated muscle cells treated with the detergent Triton X-100 had an ∼60 % greater overall activity of 5′ nucleotidase suggesting that the detergent revealed an enclosed pool of the enzyme. In this context it should be mentioned that Triton X-100 does not enhance the activity of purified 5′ nucleotidase per se as shown by Camici et al. (1985) and Stanley et al. (1980). The observation of an enclosed pool of the enzyme is in accordance with results from experiments on fibroblasts (Widnell et al. 1982), hepatocytes, lymphocytes and adipocytes (Stanley et al. 1980), showing two pools of 5′ nucleotidase, one bound to the membrane and one located in intracellular vesicles. Stanley et al. (1980) described a continuous traffic of the enzyme between the membrane and the intracellular pool of vesicles in hepatocytes. In the present study, the activity of AMP 5′ nucleotidase in non-detergent-treated homogenates (cytosolic + the ecto form of the enzyme) was significantly higher for stimulated than for non-stimulated cells. However, the total AMP 5′ nucleotidase activity (cytosolic + ecto + enclosed intracellular form), as assessed by detergent treatment of homogenates, was not different between non-stimulated and stimulated cells (Fig. 2). Furthermore, from homogenate measurements and fractionation experiments it was estimated that 48 and 34 % of the total AMP 5′ nucleotidase activity was attributed to the ecto form and the enclosed intracellular pool, respectively, in the non-stimulated cells, whereas in the stimulated cells the corresponding values were 55 and 27 %, respectively. The soluble cytosolic form constituted the same proportion (∼18 %) in non-stimulated and stimulated cells. Thus, it is plausible that the increased ecto 5′ nucleotidase activity upon muscle contraction is the result of a translocation mechanism of 5′ nucleotidase from intracellular vesicles to the membrane. The altered distribution of AMP 5′ nucleotidase upon contraction represents an approximate 15 % increase in the ecto form, which is similar to the contraction-induced increase in AMP 5′ nucleotidase in homogenates (20 %) but clearly lower than the contraction-induced increase in activity measured in intact cells (ecto form; 70 %). This difference in stimulation effect in intact cells versus cell homogenates may be explained by an alteration in distribution or activity of the enzyme upon homogenization. Further studies are needed to verify that a translocation process indeed is involved in the contraction-induced increase in the rate of adenosine formation.

Muscle contraction increases the capacity for membrane glucose transport by causing a movement of the glut 4 glucose transporter from intracellular vesicles to the cell membrane (Hirshman et al. 1988). Furthermore, in studies using adenosine receptor antagonists and adenosine deaminase in isolated rat muscle preparations the insulin- and contraction-induced increase in glucose transport has been shown to be enhanced by adenosine (Vergauwen et al. 1994; Han et al. 1998). The present data suggest that muscle contraction also induces an enhanced capacity for adenosine formation and that this improved capacity, at least in part, can be explained by a translocation process similar to that of the glut 4 transporter. It is intriguing to suggest that translocation of 5′ nucleotidase may be involved in the complex regulation of glucose uptake in skeletal muscle. This possibility should clearly be explored in the future.

Adenylate charge, defined as the mole fraction of ATP plus half the mole fraction of ADP divided by the total adenylate pool (Atkinson, 1968), has been suggested as a main regulator of 5′ nucleotidase in cardiac muscle and seminal plasma (Itoh et al. 1986; Minelli et al. 1995). In the present study, the effect of a lowering of the intracellular energy charge was examined by addition of iodoacetic acid to the cells to cause a degradation of ATP. There was a significant increase in the rate of adenosine formation with a change in the adenylate charge from approximately 0.9 to 0.4. The intracellular energy charge observed in stimulated muscle cells was similar to that of non-stimulated cells (0.89 vs. 0.92). Thus, alterations in the ATP, ADP and AMP concentrations in the intracellular environment may be of importance for the adenosine formation in vivo, but do not appear to explain the contraction-induced increase in ecto 5′ nucleotidase activity observed in the present study.

It was observed that the rate of adenosine formation was enhanced by about 18 % in non-stimulated cells that had received buffer from electrically stimulated cells. This enhancement was not due to a contraction-induced leakage of 5′ nucleotidase as no activity of 5′ nucleotidase was observed in the medium after stimulation (data not shown). A series of experiments was performed to elucidate if this enhancement was due to the effect of a metabolite released from the contracting muscle cells. Lactate, potassium, nitric oxide and ATP, which all have been found to increase in the muscle extracellular fluid upon contraction (Frandsen et al. 1998; Hellsten et al. 1998; MacLean et al. 1999), were added separately to the medium of non-stimulated muscle cells and the rate of extracellular adenosine formation was examined. The effect of an increase in intracellular calcium, an event which regulates many proteins in skeletal muscle, on the rate of adenosine formation was also investigated. This was done by the addition of terbutalin to the cells. However, none of the above-mentioned interventions affected the rate of adenosine formation by the muscle cells. The identity of the compound that apparently is released from contracting muscle and affects the rate of adenosine formation remains, thus, to be revealed.

In cardiac muscle two forms of the soluble cytosolic 5′ nucleotidase have been described, one with a greater preference for IMP than AMP and one with a greater preference for AMP (Truong et al. 1988). In the present study, the activity of the intracellular 5′ nucleotidase with IMP as substrate was about half of that with AMP as substrate, suggesting that there either may be only an AMP-preferring form(s) of the 5′ nucleotidase in skeletal muscle cells or that the AMP-preferring form is more prevalent. Furthermore, muscle contraction induced a similar relative increase in the activity of 5′ nucleotidase with IMP as with AMP as substrate. This observation implies that the contraction-induced increase in adenosine formation was not associated with an alteration in the affinity for AMP at the expense of that for IMP.

In conclusion, the present findings suggest that muscle contraction induces an enhanced potential for extracellular adenosine production by the muscle cells via an increase in the activity of the ecto form of 5′ nucleotidase. The increased 5′ nucleotidase activity may in part be induced by a yet unidentified compound released from the muscle cells and by alteration of the intracellular adenylate charge. It is also proposed that a pool of 5′ nucleotidase internalized in intracellular vesicles may contribute to the contraction induced increase in the membrane activity of 5′ nucleotidase, via a translocation mechanism. Further research is, however, needed to verify such a mechanism.

Acknowledgments

The excellent technical assistance of Karina Olsen and Merete Vannby is gratefully acknowledged. The present study was supported by the Danish Natural Research Foundation (Jnr. 504-14).

References

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