Ra adenosine receptors in human platelets. Characterization by 5'-N-ethylcarboxamido[3H]adenosine binding in relation to adenylate cyclase activity

Abstract

Adenosine receptors in human platelet membranes have been characterized by radioligand binding and measurement of adenylate cyclase activity. Binding of 5'-N-ethylcarboxamido[3H]adenosine ([3H] NECA ) was rapid, reversible and dependent on protein concentration, pH and temperature. Due to a rapid rate of dissociation (t 1/2 approximately 20 s) binding was highest at 0 degree C. Adenosine deaminase and GTP alone did not influence [3H] NECA binding, whereas several divalent cations decreased binding. Saturation experiments revealed two different binding sites for [3H] NECA , with KD values of 0.16 and 2.9 mumol/l and Bmax values of 8.4 and 33.4 pmol/mg of protein. In competition experiments NECA was the most potent adenosine agonist (IC50 0.5 mumol/l), followed by 2-chloroadenosine (IC50 6.3 mumol/l) and adenosine (IC50 12 mumol/l). A similar rank order of potencies was observed for the stimulatory effect of adenosine analogues on platelet adenylate cyclase. NECA stimulated adenylate cyclase activity with an EC50 value of 0.5 mumol/l and was approximately 4-fold more potent than (-)N6-phenylisopropyladenosine [(-)PIA]. However, (-)PIA and N6-cyclohexyladenosine did not significantly affect [3H] NECA binding, an observation not consistent with the stimulatory effect on adenylate cyclase. The adenosine antagonists 3-isobutyl-1-methylxanthine, theophylline and caffeine showed IC50 values between 98 and 5,600 mumol/l. [3H]PIA bound to platelet membranes with very low affinity and was not displaced by NECA . The [3H] NECA binding to human platelet membranes satisfies essential criteria for Ra adenosine receptors and, with some limitations, should be of value for the characterization of adenosine receptors in Ra subtype selective cells.