The effects of adenosine and adenosine analogs on adenylate cyclases from several tissues have been examined. Two adenosine-reactive sites have been identified: (i) the "R" site, occupancy of which usually leads to activation of cyclase and which requires integrity of the ribose ring for activity, and (ii) the "P" site, which mediates inhibition and requires integrity of the purine ring for activity. Biphasic effects of adenosine are explained by the presence of both sites on a single adenylate cyclase. Comparison of these data with those in the literature indicates that adenosine-reactive "P" and "R" sites are present generally.

Two distinct adenosine-sensitive sites on adenylate cyclase

Whereas adenosine itself exerted independent stimulatory and inhibitory effects on the adenylate cyclase activity of a platelet particulate fraction at low and high concentrations respectively, 2-substituted and N6-monosubstituted adenosines had stimulatory but greatly decreased inhibitory effects. Deoxyadenosines, on the other hand, had enhanced inhibitory but no stimulatory effects. The most potent inhibitors found were, in order of increasing activity, 9-(tetrahydro-2-furyl)adenine (SQ 22536), 2',5'-dideoxyadenosine and 2'-deoxyadenosine 3'-monophosphate. Kinetic studies on prostaglandin E1-activated adenylate cyclase showed that the inhibition caused by either 2',5'-dideoxyadenosine or compound SQ 22536 was non-competitive with MgATP and that the former compound, at least, showed negative co-operativity; 50% inhibition was observed with 4 micron-2',5'-dideoxyadenosine or 13 micron-SQ 22536. These two compounds also inhibited both the basal and prostaglandin E1-activated adenylate cyclase activities of intact platelets, when these were measured as the increases in cyclic [3H]AMP in platelets that had been labelled with [3H]adenine and were then incubated briefly with papaverine or papaverine and prostaglandin E1. Both compounds, but particularly 2',5'-dideoxyadenosine, markedly decreased the inhibition by prostaglandin E1 of platelet aggregation induced by ADP or [arginine]vasopressin as well as the associated increases in platelet cyclic AMP, so providing further evidence that the effects of prostaglandin E1 on platelet aggregation are mediated by cyclic AMP. 2'-Deoxyadenosine 3'-monophosphate did not affect the inhibition of aggregation by prostaglandin E1, suggesting that the site of action of deoxyadenosine derivatives on adenylate cyclase is intracellular. Neither 2',5'-dideoxyadenosine nor compound SQ 22536 alone induced platelet aggregation. Moreover, neither compound potentiated platelet aggregation or the platelet release reaction when suboptimal concentrations of ADP, [arginine]vasopressin, collagen or arachidonate were added to heparinized or citrated platelet-rich plasma in the absence of prostaglandin E1. These results show that cyclic AMP plays no significant role in the responses of platelets to aggregating agents in the absence of compounds that increase the platelet cyclic AMP concentration above the resting value.

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Inhibition of adenylate cyclase by adenosine analogues in preparations of broken and intact human platelets. Evidence for the unidirectional control of platelet function by cyclic AMP.

Adenosine may well be as important in the regulation of adenylate cyclase as hormones. Sattin and Rall first demonstrated in 1970 that adenosine was a potent stimulator of adenylate cyclase in the brain. However, adenosine is an equally potent inhibitor of adenylate cyclase in other cells such as adipocytes. The concentration of adenosine required for this regulation of adenylate cyclase is in the nanomolar range (10 to 100 nm). Both the inhibitory and stimulatory effects of low concentrations of adenosine on adenylate cyclase are antagonized by methylxanthines. This antagonism of adenosine action may account for all or part of the effects of methyl xanthines on cyclic AMP levels in many tissues. Adenosine appears to be a particularly important endogenous regulator of adenylate cyclase in brain, smooth muscle and fat cells. Under conditions in which intracellular AMP rises, adenosine formation and release is accelerated. In addition to its direct effects on adenylate cyclase, adenosine (at higher concentrations approaching millimolar) exerts multiple effects on cellular metabolism as a result of its intracellular metabolism and especially conversion to nucleotides.

R.J. Haslam

Papers

Activation and inhibition of blood platelet adenylate cyclase by adenosine or by 2-chloroadenosine.