Adenosine receptor activation by adenine nucleotides requires conversion of the nucleotides to adenosine.

Adenine nucleotides cause adenosine receptor-mediated increases in cyclic AMP in the VA13 human fibroblast line. Levels of adenosine accumulated in the medium are insufficient to account for the responses to adenine nucleotides. Since rapid conversion of the nucleotides to adenosine by 5'-nucleotidase in the vicinity of the receptor might account for the responses, six experimental methods were developed to distinguish between "local conversion" and direct action of the nucleotides. Results of all six methods favored local conversion. (1)5'-Nucleotidase inhibitors blocked the accumulations of cyclic AMP elicited by AMP, ADP, and ATP, but did not affect the response to adenosine. The most potent inhibitor of both conversion of AMP and response to AMP was alpha, beta-methylene-ADP (APCP). (2) Adenosine deaminase blocked the responses to AMP, ADP, ATP, and adenosine-containing coenzymes. (3) Theophylline, a specific competitive adenosine antagonist, was an insurmountable inhibitor of the increases in cyclic AMP caused by AMP, ADP, and ATP. The insurmountability was presumably due to substrate saturation of the converting enzyme 5'-nucleotidase. (4) Although ADP and ATP had partial agonist-liked dose-response curves, they did not inhibit the response to adenosine. (5) Nine cell lines which responded to adenosine were tested for response to AMP. Cell lines with high levels of 5'-nucleotidase had large responses to AMP, those with intermediate levels of 5'-nucleotidase had large or intermediate responses to AMP, and those with low 5'-nucleotidase levels did not respond to AMP. (6) Inhibition of the uptake of labelled adenosine was used as an indicator of unlabelled adenosine concentrations near the cell membrane. Unlabelled AMP inhibited uptake nearly as effectively as unlabelled adenosine. APCP reversed the inhibition by AMP but not the inhibition by adenosine. The adenosine receptor is concluded to be an entity distinct from adenine nucleotide receptors.