Molecular Pharmacology, Vol 11, 528-544, Copyright © 1975 by the American Society for Pharmacology and Experimental Therapeutics

Effects of Adenosine on Levels of Adenosine Cyclic 3',5'-Monophosphate in Human Blood Platelets in Relation to Adenosine Incorporation and Platelet Aggregation

¹ Department of Pathology, McMaster University, Hamilton, Ontario, Canada L8S 4J9

Cyclic 3', 5'-[¹⁴C]AMP was measured in platelets that had first been incubated with [¹⁴C]adenine. Maximum increases of 2-4-fold were observed 0.5 min after addition of 10-40 µM adenosine. Smaller increases were obtained with higher concentrations of adenosine. In 0.5-min incubations 2-chloroadenosine was less effective than adenosine at concentrations below 20 µM and more effective at concentrations above 100 µM. Incorporation of 1-10 µM adenosine into platelets was inhibited at least 96% by p-nitrobenzylthioguanosine without any effect on the increase in cyclic [¹⁴C]AMP caused by these concentrations of adenosine, suggesting that adenosine acts at an extracellular site. With higher adenosine concentrations, p-nitrobenzylthioguanosine was less effective in inhibiting incorporation of adenosine but blocked the decline in cyclic [¹⁴C]AMP levels observed on increasing the adenosine concentration above 40 µM. This inhibitory effect of high adenosine concentrations on the accumulation of cyclic [¹⁴C]AMP was more easily detected when adenosine was added with prostaglandin E₁ and represents a second, possibly intracellular action of adenosine unrelated to its effect in increasing cyclic AMP levels. Papaverine markedly potentiated the increase in platelet cyclic [¹⁴C]AMP observed with all concentrations of adenosine, indicating that adenosine activates platelet adenylate cyclase. The kinetics of this activation were studied in intact platelets incubated for short intervals in the presence of papaverine. Adenosine (K _A = 1 µM) activated platelet adenylate cyclase up to a maximum of 8-10-fold. This action of adenosine was competitively inhibited by caffeine (K_i = 72 µM) or theophylline (K_i = 25 µM). No inhibitory effect of high adenosine concentrations on cyclic [¹⁴C]AMP formation was observed in intact platelets in the presence of papaverine. The plateletaggregating agents ADP and epinephrine, but not vasopressin, markedly inhibited the increase in platelet cyclic [¹⁴C]AMP with adenosine. ADP was found to be a noncompetitive inhibitor (K_i = 0.9 µM) of the effect of adenosine on adenylate cyclase in intact platelets. Some close correlations were observed between the effects of adenosine on platelet cyclic [¹⁴C]AMP levels and on platelet aggregation. Caffeine partially blocked the inhibition of aggregation by adenosine. As a whole the results show that platelets possess a specific extracellular membrane receptor for adenosine, which is distinct from that for ADP and which mediates the inhibition of platelet function by adenosine by activating platelet adenylate cyclase.

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