The influence of hypoxia on intracellular pH of isolated fibres from rat soleus muscle

Abstract

We have previously found that resting dog skeletal muscle released small amounts of both adenosine and lactate. Systemic hypoxia elevated arterial lactate, leading to lactate uptake into the muscle, and elevated adenosine output. Treatment with a-cyano-4-hydroxycinnamic acid (CHCA) prevented the lactate uptake during hypoxia, and abolished the adenosine output (Mo & Ballard, 1993), which suggested that the increased adenosine output in systemic hypoxia was mediated by intracellular pH depression. The present experiments were undertaken to investigate further the abolition of the resting adenosine output by hypoxia in the presence of CHCA. A bundle of around fifteen fibres was mechanically dissected from rat soleus muscle, mounted in the perfusion chamber in a Hepes-buffered, oxygenated medium, and left to stabilize for at least 30 min. The tissue was then loaded with the methyl ester of 2',7'-bis-(2-carboxyethyl)-5- (and-6) -carboxyfluorescein (BCECF, 5#M, a fluorescent pH indicator) for 30 min at 37 0, after which the tissue was washed for 10 min. Intracellular pH was determined from the emissions at 520 nm, with an excitation ratio of 499 to 440 nm. At a P02 of 415-4 + 20-2 mmHg, the extracellular pH was 7-28 + 0 04 and the intracellular pH was 6-95 + 0 09 (mean + S.E.M., n = 8). After 10 min exposure to a P02 of 54-5 + 3-5 mmHg, the extracellular pH remained at 7 30 + 0 04, but the intracellular pH increased to 7 79 + 0-12 (P < 0 05, paired t test). CHCA did not significantly alter the relationship between Po2 and intracellular pH; at a P02 of 464-8 + 17 2 mmHg, the intracellular pH was 7-15 + 0-08, and at a Po2 of 54-2 + 7-2 mmHg, the intracellular pH was 7-80 + 0-15 mmHg (n = 5). However, addition of 5 mm lactic acid to the perfusate (a similar concentration to that seen in arterial blood in systemic hypoxia) depressed the extracellular pH from 7 39 + 0 01 to 7-27 + 0.01 and the intracellular pH from 7-07 + 0-06 to 6-89 + 0-06 (n = 6; P < 0 05, paired t test). We conclude that the direct effect of hypoxia on red skeletal muscle is to raise the intracellular pH, and that this may be responsible for a reduction in adenosine output. However, systemic hypoxia probably depresses the intracellular pH of the muscle due to increased lactate uptake, which may explain the increased adenosine output in that condition. REFERENCE Mo, F.M. & Ballard, H.J. (1993). Proc. Int. Union Physiol. Sci. 18, 173.