The effects of pH depression on the adenosine output from the in-situ buffer-perfused rat gracilis muscle

Abstract

In-vivo studies showed that adenosine output increased with the depression of pH during either muscle contractions or acid infusion. However, the effect of pH elevation on adenosine output has never been reported. Several enzymes may be involved in the adenosine production from AMP, namely 5’-nucleotidase and the non-specific phosphatases, including alkaline phosphatase. The contribution of these enzymes to the adenosine production may vary at different pH values. We tested the effect of depression or elevation of pH on the adenosine output from rat gracilis muscle. The vascularly-isolated muscle was perfused in-situ at a constant flow rate of 40-50 pl/min/g. The pH of the perfusion buffer (Kreb’s-bicarbonate equilibrated against 95% 02 / 5% COZ, 37°C) was either depressed to 6.8 or elevated to 7.8 by substituting 30 mM sodium chloride with a similar concentration of sodium acetate or ammonium chloride, respectively. The vascularly isolated muscle was stretched to its resting length (1,) and perfused via a muscular branch of the femoral artery with buffer at pH 7.4 (control) and thereafter with buffer either at pH 6.8 (acidosis) or at pH 7.8 (alkalosis). Samples of venous effluent collected during both control and acidosis or alkalosis were purified by organic extraction and their adenosine concentrations were determined by reversed-phase high-performance liquid chromatography. Acidosis increased the venous adenosine concentration from 784 + 167 to 1170 + 263 nM (n=6; PcO.02 Student’s t-test); however, alkalosis did not significantly alter the venous adenosine (595 f 3 18 nM in control and 763 + 191 nM in alkalosis). Adenosine formation by 5’-nucleotidase increases at low pH, which may account for the enhanced adenosine production in acidosis. At elevated pH the adenosine formation by 5’- nucleotidase decreases, whereas the formation by alkaline phosphatase increases. These data suggest that the overall rate of adenosine production does not change with moderate elevation of pH. We speculate that the relative contributions of 5’-nucleotidase and alkaline phosphatase to the adenosine production may change as the pH increases.