A possible mechanism for the control of adenosine formation by changes in pH in rat skeletal muscle

Abstract

Under in-vivo conditions, adenosine output from oxidative skeletal muscle is stimulated by a decrease in pH. The present experiments were undertaken to investigate the enzymatic control of adenosine formation by changes in pH. The membrane and cytosolic fractions of rat oxidative muscle tissues (soleus) were separated by differential centrifugation. The V,, and K, of the adenosineforming enzyme 5’-nucleotidase (ND) were determined at pH values of 6.5, 7.0, 7.5 and 9.0; the rate of adenosine formation was estimated from these data and literature value for the substrate concentration. For the membrane bound (ecto-) ND, the highest V max was found at pH 7.5, which was 1.7, 1.2 and 1.9 times that at pH 6.5, 7.0 and 9.0, respectively, whereas the affinity (reciprocal of K,) of ecto-ND for AMP was decreased as the pH was increased from 6.5 to 9.0. Therefore, the estimated rates of adenosine formation (nmol/min/g wet. wt.) by ecto-ND were 50.4 at pH 6.5, 48.5 at pH 7.0, 37.0 at pH 7.5 and 19.7 at pH 9.0, respectively. For the soluble (cytosolic) ND, the V,, was increased as the pH was increased from 6.5 to 9.0, but the affinity of cytosolic ND for AMP was similar under all conditions. The estimated rate of adenosine formation by cytosolic ND did not exceed 0.7 nmol/min/g wet. wt. at any pH. These results suggest that in oxidative skeletal muscle (1) the increased adenosine formation during pH depression is the result of the increased affinity (decreased K, value) of the ecto-ND for the substrate (AMP) and (2) the rate of adenosine formation by the membrane bound ecto-ND is sufficient to account for the adenosine release observed under in-vivo conditions such as muscle contractions. On the other hand, the V,, and K, of the cytosolic ND indicate that it is unlikely to account for adenosine formation in skeletal muscle in-vivo.