Abstract
A flexibly mounted microdialysis probe was used to measure the concentration of interstitial adenosine in in vivo rat hearts. The level of adenosine during perfusion of adenosine 5'-monophosphate (AMP) was given as an index of the activity of ecto-5'-nucleotidase in the tissue. The adenosine dialysate measured the concentration using reversed-phase high performance liquid chromatography (HPLC) and the absorbance of the column eluate was monitored at 260 nm using an ultraviolet detector. The baseline level of adenosine concentration measured in the absence of exogenous AMP was -0.5 μM, which was lower than the level of adenosine concentration seen in the presence of 100 μM AMP (-9 μM). In the presence of the selective inhibitor of ecto-5'-nucleotidase, α, β-methyleneadenosine-5'-diphosphate (AOPCP) at a concentration of 100μM in the perfusate, AMP (100 μM) -induced increases of adenosine in the dialysate were completely inhibited and remained at -0.5 μM, a level close to the baseline. When various concentrations of AMP (ranging from 10 to 1000μM) were applied through the probe, the steady state levels of dialysate adenosine rose with increases in the concentration of AMP. The EC50 values was 107.2 μM. This value is much closer to the Km for 'ecto-'than that for 'cytosolic'5'-nucleotidase. Ecto-5'-nucleotidase has a lower Km for AMP (-20 μM) than does cytosolic 5'-nucleotidase (-3 mM). Under a constant supply of substrate (AMP) a limiting factor for the production of adenosine would be the activity of the catalytic enzyme for this reaction, namely ecto-5'-nucleotidase.
