Evidence for control of adenosine metabolism in rat oxidative skeletal muscle by changes in pH

1. We investigated the effects of pH elevation or depression on adenosine o utput from buffer-perfused rat gracilis muscle, and kinetic properties of a denosine-forming enzymes, 5'-nucleotidase (5'N) and non-specific phosphatas e (PT), and adenosine-removing enzymes, adenosine kinase (AK) and adenosine deaminase (AD), in homogenates of muscle. 2. Depression of the perfusion buffer pH from 7.4 to 6.8, by addition of so dium acetate, reduced arterial perfusion pressure from 8.44 +/- 1.44 to 7.3 3 +/- 0.58 kPa, and increased adenosine output from 35 +/- 5 to 56 +/- 6 pm ol min(-1) (g wet wt muscle)(-1) and AMP output from 1.8 +/- 0.3 to 9.1 +/- 3.9 pmol min(-1) (g wet wt muscle)(-1) 3. Elevation of the buffer pH to 7.8, by addition of ammonium chloride, red uced arterial perfusion pressure from 8.74 +/- 0.57 to 6.96 +/- 1.37 kPa, a nd increased adenosine output from 25 +/- 5 to 47 +/- 8 pmol min(-1) (g wet wt muscle)(-1) and AMP output from 3.7 +/- 1.1 to 24.6 +/- 6.8 pmol min(-1 ) (g wet wt muscle)(-1) 4. Activity of membrane-bound 5'N was an order of magnitude higher than tha t of either cytosolic 5'N or PT: pH depression reduced the K-m of 5'N, whic h increased its capacity to form adenosine bg 10-20% for every 0 5 unit dec rease in pH wit hin the physiological range. PT was only found in the membr ane fraction: its contribution to extracellular adenosine formation increas ed from about 5% at pH 7.0 to about 15% at pH 8 0. 5. Cytosolic 5'N had a low activity, which was unaffected by pH; the rate o f intracellular adenosine formation was an order of magnitude lower than th e rate of adenosine removal by adenosine kinase or adenosine deaminase, whi ch were both exclusively intracellular enzymes. 6. We conclude that (i) adenosine is formed in the extracellular compartmen t of rat skeletal muscle, principally by membrane-bound 5'N, where it is pr otected from enzymatic breakdown; (ii) adenosine is formed intracellularly at a very low rate, and is unlikely to leave the cell; (iii) enhanced adeno sine formation at low pH is driven by an increased extracellular AMP concen tration and an increased affinity of membrane-bound 5'N for AMP; (iv) enhan ced adenosine formation at high pH is driven solely by the elevated extrace llular AMP concentration, since the catalytic capacity of membrane 5'N is r educed at high pH.