Concentrative transport of adenosine in murine splenocytes: limitation by an ecto-adenosine deaminase

Abstract

Coincident with studies of the transport of (3H]adenosine in murine splenocytes, we have evidence for the extracellular degradation of adenosine. A Na(+)-dependent active transport system for nucleosides exists in splenocytes, but no intracellular concentration gradient of adenosine was observed. Inhibition of adenosine transport across the plasma membrane by dipyridamole and a Na(+)-free medium did not prevent the deamination of extracellular adenosine by what has been generally considered to be a cytosolic enzyme. This failure to achieve an adenosine concentration gradient appears to be consequent to the action of a very active ecto-adenosine deaminase. Inhibition of the adenosine deaminase by deoxycoformycin permits a 6-fold increase in intracellular adenosine concentration relative to the medium by the Na(+)-dependent process. Rapid inhibition of adenosine deaminase by deoxycoformycin occurs even in the presence of dipyridamole which prevents the entry of deoxycoformycin as well as adenosine into the cells in a Na(+)-free medium. These results further support the view that this is an ectoenzyme activity. The kinetics of active adenosine transport were Km = 7.8 +/- 1.1 microM with Vmax = 8.2 +/- 2.8 microM/s in a Na+ medium and much less efficiently in a Li+ medium (Km = 250 +/- 50 microM,Vmax = 7.8 +/- 1.3 microM/s). Inhibition of adenosine transport by other nucleosides suggests a single Na(+)-dependent nucleoside transport system in murine splenocytes with narrow substrate specificity.