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 output from buffer-perfused rat gracilis muscle, and kinetic properties of adenosine-forming enzymes, 5'-nucleotidase (5'N) and non-specific phosphatase (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 sodium acetate, reduced arterial perfusion pressure from 8.44 +/- 1.44 to 7.33 +/- 0.58 kPa, and increased adenosine output from 35 +/- 5 to 56 +/- 6 pmol 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, reduced arterial perfusion pressure from 8.74 +/- 0.57 to 6.96 +/- 1.37 kPa, and 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 that of either cytosolic 5'N or PT: pH depression reduced the K(m) of 5'N, which increased its capacity to form adenosine by 10-20% for every 0.5 unit decrease inpH within the physiological range. PT was only found in the membrane fraction: its contribution to extracellular adenosine formation increased 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 of intracellular adenosine formation was an order of magnitude lower than the rate of adenosine removal by adenosine kinase or adenosine deaminase, which were both exclusively intracellular enzymes. 6. We conclude that (i) adenosine is formed in the extracellular compartment of rat skeletal muscle, principally by membrane-bound 5'N, where it is protected from enzymatic breakdown; (ii) adenosine is formed intracellularly at a very low rate, and is unlikely to leave the cell; (iii) enhanced adenosine formation at low pH is driven by an increased extracellular AMP concentration and an increased affinity of membrane-bound 5'N for AMP; (iv) enhanced adenosine formation at high pH is driven solely by the elevated extracellular AMP concentration, since the catalytic capacity of membrane 5'N is reduced at high pH.