1. We investigated the effect of moderate systemic hypoxia on the arterial, venous and interstital concentration of adenosine and adenine nucleotides in the neurally and vascularly isolated, constant-flow perfused gracilis muscles of anaesthetized dogs.2. Systemic hypoxia reduced arterial P O 2 from 129 to 28 mmHg, venous P O 2 from 63 to 23 mmHg, arterial pH from 7.43 to 7.36 and venous pH from 7.38 to 7.32. Neither arterial nor venous P CO 2 were changed. Arterial perfusion pressure remained at 109 ± 8 mmHg for the first 5 min of hypoxia, then increased to 131 ± 11 mmHg by 9 min, and then decreased again throughout the rest of the hypoxic period.3. Arterial adenosine (427 ± 98 nM) did not change during hypoxia, but venous adenosine increased from 350 ± 52 to 518 ± 107 nM. Interstitial adenosine concentration did not increase (339 ± 154 nM in normoxia and 262 ± 97 nM in hypoxia). Neither arterial nor venous nor interstitial concentrations of adenine nucleotides changed significantly in hypoxia.4. Interstitial adenosine, AMP, ADP and ATP increased from 194 ± 40, 351 ± 19, 52 ± 7 and 113 ± 36 to 764 ± 140, 793 ± 119, 403 ± 67 and 574 ± 122 nM, respectively, during 2 Hz muscle contractions.5. Adenosine, AMP, ADP and ATP infused into the arterial blood did not elevate the interstitial concentration until the arterial concentration exceeded 10 µM.6. We conclude that the increased adenosine in skeletal muscle during systemic hypoxia is formed by the vascular tissue or the blood cells, and that adenosine is formed intracellularly by these tissues. On the other hand, adenosine formation takes place extracellularly in the interstitial space during muscle contractions.

Single fibres were dissected from mouse flexor digitorum brevis muscles and subjected to a protocol of eccentric stretches consisting of ten tetani each with a 40 % stretch. Ten minutes later the fibres showed a reduced force, a shift in the peak of the force-length relation and a steepening of the force-frequency relation. Addition of the fluorescent dye sulforhodamine B to the extracellular space enabled the T-tubular system to be visualized. In unstimulated fibres and fibres subjected to 10 isometric tetani, the T-tubules were clearly delineated. Sulforhodamine B diffused out of the T-tubules with a half-time of 18 ± 1 s. Following the eccentric protocol, vacuoles connected to the T-tubules were detected in six out of seven fibres. Sulforhodamine B diffused out of the vacuoles of eccentrically damaged fibres extremely slowly with a half-time of 6.3 ± 2.4 min and diffused out of the T-tubules with a half-time of 39 ± 4 s. Vacuole production was eliminated by application of 1 m ouabain to the muscle during the eccentric protocol. On removal of the ouabain, vacuoles appeared over a period of 1 h and were more numerous and more widely distributed than in the absence of ouabain. We propose that T-tubules are liable to rupture during eccentric contraction probably because of the relative movement associated with the inhomogeneity of sarcomere lengths. Such rupture raises intracellular sodium and when the sodium is pumped from the cell by the sodium pump, the volume load of Na + and water exceeds the capacity of the T-tubules and causes vacuole production. The damage to the T-tubules may underlie a number of the functional changes that occur in eccentrically damaged muscle fibres.

The effect of eccentric contractions on intracellular Na(+) concentration ([Na(+)](i)) and its distribution were examined in isolated rat and mouse muscle fiber bundles. [Na(+)](i) was measured with either Na(+)-binding benzofuran isophthalate or sodium green. Ten isometric contractions had no significant effect on force (measured after 5 min of recovery) and caused no significant change in the resting [Na(+)](i) (7.2 +/- 0.5 mM). In contrast 10 eccentric contractions (40% stretch at 4 muscle lengths/s) reduced developed force at 100 Hz to 45 +/- 3% of control and increased [Na(+)](i) to 16.3 +/- 1.6 mM (n = 6; P < 0.001). The rise of [Na(+)](i) occurred over 1-2 min and showed only minimal recovery after 30 min. Confocal images of the distribution of [Na(+)](i) showed a spatially uniform distribution both at rest and after eccentric contractions. Gd(3+) (20 microM) had no effect on resting [Na(+)](i) or control tetanic force but prevented the rise of [Na(+)](i) and reduced the force deficit after eccentric damage. These data suggest that Na(+) entry after eccentric contractions may occur principally through stretch-sensitive channels.

SUMMARY1. In dogs anaesthetized with sodium pentobarbitone and artificially ventilated, the gracilis muscles were vascularly isolated and perfused at a constant flow rate of 51-2+9-8 ml min-1 100 g-1 muscle tissue (183+ 17-8% of resting blood flow; mean+S.E.; n = 13).2. Electrical stimulation of the cut peripheral end of the obturator nerve (6 V, 4 Hz) resulted in muscle contraction (658+118 g 100 g-1 force after 5 min), and an immediate decrease in arterial perfusion pressure from 179+15-7 mmHg to 87+10-0 mmHg (514+4-5 % decrease in vascular resistance after 2 min of contraction). Venous oxygen tension decreased from 692 + 51 mmHg to 18-5 + 1-4 mmHg (n = 6). These values did not significantly alter during the remaining period of stimulation (10-20 min).3. The concentration of adenosine in arterial plasma did not change significantly during muscle contraction (137 + 23 nM; n = 10). However, the adenosine concentrations in venous plasma showed a significant (P < 0-01) increase from a control value of 164 + 55 nM to 455+77 nM (n = 9) after 5 min of muscle contraction and remained high during the rest of the 20 min contraction. In six of the dogs adenosine concentrations were determined after 1 and 3 min of contraction and showed a smaller but statistically significant (P < 0 05) rise in venous concentration.4. During infusion of adenosine into the artery to give plasma concentrations between 0-3 /LM and 1 mm, 72-6 + 2-1 % (n = 29) of the infused adenosine was taken up by the tissues before it reached the vein. Comparison of vasodilatation and venous adenosine concentrations during adenosine infusion and muscle contractions showed that the released adenosine could contribute about 15 % to the total vasodilatation after 1 min and about 40 % between 5 and 20 min of contractions. Released adenosine could contribute about 80% to the vasodilatation that remained 5 min after the withdrawal of stimulation. Arterial perfusion pressure took 22 min to return to control, whereas adenosine release had fallen to zero within 10 min.5. These data suggest that the released adenosine could contribute to exercise hyperaemia, but is unlikely to be the main factor, particularly in the initial stage.

The present study was performed to investigate the effect of acidosis on the efflux of ATP from skeletal muscle. Infusion of lactic acid to the perfused hindlimb muscles of anaesthetised rats produced dose-dependent decreases in pH and increases in the interstitial ATP of extensor digitorum longus (EDL) muscle: 10 mm lactic acid reduced the venous pH from 7.22 ± 0.04 to 6.97 ± 0.02 and increased interstitial ATP from 38 ± 8 to 67 ± 11 nm. The increase in interstitial ATP was well-correlated with the decrease in pH (r 2 = 0.93; P < 0.05). Blockade of cellular uptake of lactic acid using α-cyano-hydroxycinnamic acid abolished the lactic acid-induced ATP release, whilst infusion of sodium lactate failed to depress pH or increase interstitial ATP, suggesting that intracellular pH depression, rather than lactate, stimulated the ATP efflux. Incubation of cultured skeletal myoblasts with 10 mm lactic acid significantly increased the accumulation of ATP in the bathing medium from 0.46 ± 0.06 to 0.76 ± 0.08 μm, confirming the skeletal muscle cells as the source of the released ATP. Acidosis-induced ATP efflux from the perfused muscle was abolished by CFTR inh -172, a specific inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), or glibenclamide, an inhibitor of both K ATP channels and CFTR, but it was not affected by atractyloside, an inhibitor of the mitochondrial ATP transporter. Silencing of the CFTR gene using an siRNA abolished the acidosis-induced increase in ATP release from cultured myoblasts. CFTR expression on skeletal muscle cells was confirmed using immunostaining in the intact muscle and Western blotting in the cultured cells. These data suggest that depression of the intracellular pH of skeletal muscle cells stimulates ATP efflux, and that CFTR plays an important role in the release mechanism.