METHOTREXATE AND SULFASALAZINE PROMOTE ADENOSINE RELEASE BY A MECHANISM THAT REQUIRES ECTO-5'-NUCLEOTIDASE-MEDIATED CONVERSION OF ADENINE-NUCLEOTIDES

We and others have shown that an increased extracellular concentration of adenosine mediates the antiinflammatory effects of methotrexate an d sulfasalazine both in vitro and in vivo, but the mechanism by which these drugs increase extracellular adenosine remains unclear. The resu lts of the experiments reported here provide three distinct lines of e vidence that adenosine results from the ecto-5'-nucleotidase-mediated conversion of adenine nucleotides to adenosine. First, pretreatment of a human microvascular endothelial cell line (HMEC-1) with methotrexat e increases extracellular adenosine after exposure of the pretreated c ells to activated neutrophils; the ecto-5'-nucleotidase inhibitor alph a,beta-methylene adenosine-5'-diphosphate (APCP) abrogates completely the increase in extracellular adenosine. Second, there is no methotrex ate-mediated increase in extracellular adenosine concentration in the supernate of cells deficient in ecto-5'-nucleotidase, but there is a m arked increase in extracellular adenosine concentration in the superna tes of these cells after transfection and surface expression of the en zyme. Finally, as we have shown previously, adenosine mediates the ant iinflammatory effects of methotrexate and sulfasalazine in the murine air pouch model of inflammation, and injection of APCP, the ecto-5'-nu cleotidase inhibitor, abrogates completely the increase in adenosine a nd the decrement in inflammation in this in vivo model. These results not only show that ecto-5'-nucleotidase activity is a critical mediato r of methotrexate-and sulfasalazine-induced antiinflammatory activity in vitro and in vivo but also indicate that adenine nucleotides, relea sed from cells, are the source of extracellular adenosine.