[ 3 H]‐SCH 58261 labelling of functional A 2A adenosine receptors in human neutrophil membranes

The present study describes the direct labelling of A 2A adenosine receptors in human neutrophil membranes with the potent and selective antagonist radioligand, [ 3 H]‐5‐amino‐7‐(2‐phenylethyl)‐2‐(2‐furyl)‐pyrazolo[4,3‐e]‐1,2,4 triazolo[1,5‐c]pyrimidine, ([ 3 H]‐SCH 58261). In addition, both receptor affinity and potency of a number of adenosine receptor agonists and antagonists were determined in binding, adenylyl cyclase and superoxide anion production assays. Saturation experiments revealed a single class of binding sites with K d and B max values of 1.34 n M and 75 fmol mg −1 protein, respectively. Adenosine receptor ligands competed for the binding of 1 n M [ 3 H]‐SCH 58261 to human neutrophil membranes, with a rank order of potency consistent with that typically found for interactions with the A 2A adenosine receptors. In the adenylyl cyclase and in the superoxide anion production assays the same compounds exhibited a rank order of potency identical to that observed in binding experiments. Thermodynamic data indicated that [ 3 H]‐SCH 58261 binding to human neutrophils is entropy and enthalpy‐driven. This finding is in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A 2A adenosine receptors. It was concluded that in human neutrophil membranes, [ 3 H]‐SCH 58261 directly labels binding sites with pharmacological properties similar to those of A 2A adenosine receptors of other tissues. The receptors labelled by [ 3 H]‐SCH 58261 mediated the effects of adenosine and adenosine receptor agonists to stimulate cyclic AMP accumulation and inhibition of superoxide anion production in human neutrophils.British Journal of Pharmacology (1998) 123 , 1723–1731; doi: 10.1038/sj.bjp.0701758