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 if adenosine deaminase by deoxycoformation occurs even in the presence of dipyridamole which prevents the entry of deoxycoformycin as well as adenosine into the cells in the Na+-free medium. These results further support the view that this is an ectoenzyme activity. The kinetics active adenosine transport were Km = 7.8 ± 1.1 μM with Vmax = 8.2 ± 2.8 μM/s in a Na+ medium and much less efficiently in a Li+ medium (Km= 250 ± 50 μM, Vmax = 7.8 ± 1.3 μM/s). Inhibition of adenosine transport by other nucleosides suggest a single Na+-dependent nucleoside transport system in murine splenocytes with narrow substrate specificity.