Regulation of Adenosine 3′:5′-Monophosphate Content in Human Astrocytoma Cells by Adenosine and the Adenine Nucleotides

Abstract

Abstract Adenosine and the adenine nucleotides AMP, ADP, and ATP (but not adenine) cause a rapid increase in the concentration of adenosine 3':5'-monophosphate (cAMP) in cultured human astrocytoma cells. Adenosine and AMP are equi-potent, causing a half-maximal rise in cAMP content at a concentration of about 10 µm. The time course of a rise in cAMP content to the maximal level effected by adenosine or AMP is the same (t1/2 ≃ 1 min). AMP is converted to adenosine in the incubation medium but not to an extent sufficient to account for its effect. The rate of uptake of adenosine into the cells is markedly faster than that of AMP, even though the t1/2 of their effects is the same. During a 10-min exposure of cells to 100 µm [3H]adenosine, the intracellular concentration of cAMP rises to 600 to 800 pmoles per mg of protein, of which only 30 to 40 pmoles per mg of protein are contributed by exogenous adenosine. The uptake of adenosine can be inhibited to greater than 95% by dipyridamole without any inhibition of the effect of adenosine on cAMP content. Such observations suggest that nucleotides act directly and need not be converted to adenosine to cause a rise in intracellular cAMP and that adenosine need not enter the cell to increase cAMP content. Theophylline is a rapidly acting, competitive inhibitor of the effect of adenosine. The inhibitory effect of theophylline is presumed to occur at the proposed extracellular site of action of adenosine and could not be related by our experiments to its well known action as an inhibitor of phosphodiesterase activity. On the other hand, papaverine potentiates the effect of adenosine, presumably as a result of the inhibition of phosphodiesterase. In contrast to theophylline, papaverine is a potent inhibitor (I50 = 17 µm) of the soluble, low Km phosphodiesterase from the astrocytoma cells. Catecholamines, which potentiate the effect of adenosine in brain slices, cause only additive increases in cAMP content in the astrocytoma cells when present with adenosine. Neither α- nor β-adrenergic receptor antagonists (phentolamine and propranolol) have any inhibitory action on the effect of adenosine. The results suggest that adenosine increases intracellular cAMP content as a result of its interaction with a specific plasma membrane surface receptor which results in the activation of adenylate cyclase.