Adenosine transport by primary cultures of neurons from chick embryo brain

Abstract

The transport of adenosine was studied in pure cultures of neurons from chick embryo brain. In order to avoid complications due to adenosine metabolism, the cells were depleted of ATP by treatment with cyanide and iodoacetate prior to incubation with [3H]adenosine. During the 5-25-s periods used for transport assays, no significant adenosine metabolism was detectable. ATP depletion reduced the initial rate of adenosine entry by less than 10%, but blocked over 90% of the radioactivity accumulated by untreated cells after 15 min. Elimination of sodium or chloride from the uptake medium had no effect on adenosine transport activity. The kinetics of adenosine entry into ATP depleted neurons obeyed the Michaelis-Menten relationship and yielded a Km of 13 microM and Vmax of 0.15 nmol/min/mg protein. The neuronal transport system has apparent selectivity for adenosine, since thymidine, inosine, or guanosine gave significant inhibition only at levels 10-100-fold higher than [3H]adenosine. Adenosine derivatives (N6-cyclohexyl-, N6-benzyl-, N6-methyl-, and 2-chloroadenosine) were more effective inhibitors; p-nitrobenzylthioinosine and dipyridamole were the most potent compounds found. These results describe a high-affinity, facilitated diffusion system for adenosine in cerebral neurons, which could participate in terminating regulatory actions of this compound in the nervous system.