Ecto-5'-nucleotidase does not catalyze vectorial production of adenosine in the perfused rat liver.
From the results obtained in perfused rat hearts, Frick and Lowenstein proposed that ecto-5'-nucleotidase catalyzes a vectorial reaction in which AMP hydrolysis is accompanied by transfer of adenosine across the cell membrane (Frick, G.P., and Lowenstein, J.M. (1978) J. Biol. Chem. 253, 1240-1244). We have examined by the use of perfused rat livers, the uptake mechanism of adenosine generated from AMP by ecto-5'-nucleotidase. Recirculating perfusion of rat livers was performed with a buffered saline containing [2-3H]AMP at an initial concentration of 5 microM. One-half of the [2-3H] AMP was dephosphorylated by 0.8 min of perfusion; less than 13% of the radioactivity of hydrolyzed [2-3H] AMP was located in [2-3H]adenosine plus [2-3H]inosine appearing in the perfusate. Addition of 6-[(4-nitrobenzyl)thio]-guanosine, an inhibitor of the nucleoside transport system, at 120 microM to the perfusate caused a 3.9-fold increase in the amount of the [2-3H]AMP-derived 3H-nucleosides appearing in the perfusate. Moreover, in a perfusion in which uridine was added to the perfusate at 2.6 mM to compete with the [2-3H] AMP-derived [2-3H]adenosine for the nucleoside transport system, more than 87% of the radioactivity of hydrolyzed [2-3H]AMP was located in [2-3H]adenosine appearing in the perfusate; the result indicates that the uridine added to the perfusate efficiently trapped the [2-3H]adenosine formed from [2-3H]AMP. These results support an uptake mechanism of the AMP-derived adenosine in which the adenosine formed by ecto-5'-nucleotidase in the blood-sinusoidal plasma membrane of hepatocytes, is taken up by the nucleoside transport system located at the plasma membrane side by side with ecto-5'-nucleotidase. The results therefore indicate that the ecto-5'-nucleotidase of hepatocytes does not catalyze vectorial production of adenosine, in contrast to the previous report on perfused rat hearts.
D015720:5'-Nucleotidase; D000241:Adenosine / Q000096:biosynthesis*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D002250:Carbon Radioisotopes; D000242:Cyclic AMP / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D009708:Nucleotidases / Q000378:metabolism*; D010477:Perfusion; D051381:Rats; D011919:Rats, Inbred Strains; D014316:Tritium
D002250:Carbon Radioisotopes; D014316:Tritium; D000249:Adenosine Monophosphate; D000242:Cyclic AMP; D009708:Nucleotidases; D015720:5'-Nucleotidase; D000241:Adenosine